o 



TORY DfACN^SIS 



ER AND UNCOLN 




Class JLS^lZ 
Book - h <d_ 



CDPXRIGHT DEPOSIT. 



MANUAL 

Of 

Laboratory Diagnosis 



By 

STELLA M. GARDNER, M. D. 

// 

and 



MARY C. 'LINCOLN, Ph. B., M. D. 

Formerly Assistant Professors of Laboratory Diagnosis, College of 
Medicine, University of Illinois. 



Chicago Medical Book Company 

Chicago 

1917 



<\ 



«& 



Copysiyht, 1917, 
CHICAGO MEDICAL BOOK CO. 




©CI.A478857 



PREFACE. 

The material in this manual has as its basis the lecture 
notes and outlines prepared by us and used during the past 
eight years in teaching the subject of Laboratory Diag- 
nosis. 

It has been our purpose first and chiefly to give practical 
working directions for making the important clinical labo- 
ratory tests, and, second, to give the clinical significance of 
the findings. 

It is published in the hope that it may be of service to 
students of medicine and to physicians who do their own 
laboratory work or who want assistance in the interpreta- 
tion of laboratory findings. It is believed, also, that it may 
be found useful to trained nurses and to technicians in 
clinical laboratories. 

STELLA M. GARDNER, M. D. 
MARY C. LINCOLN, M. D. 

Chicago. 



TABLE OF CONTENTS 



Chapter I. 
Blood 



Obtaining the Blood for Examination. 
Estimation of Haemoglobin. 
Counting the Corpuscles. 
Making and Staining Blood Films. 
Differential Leucocyte Counting. 
Coagulation Time. 
General Pathology of the Blood. 
Diseases of the Blood. 

Chapter II. 
Serum Diagnosis ■ 29 

Widal Test for Typhoid. 

Wassermann Test for Syphilis. 

Complement Fixation Test for Gonorrhoea. 

Chapter III. 
Urine : 43 

Physical Examination. 

Chemical Examination. 

Sediments. 

Table of Diagnostic Urinary Findings in Certain Diseases. 

Chapter IV. 
Stomach Contents 69 

Test Meals. 

Macroscopic Examination, 



TABLE OF CONTENTS 

Chemical Examination. 

Normal Stomach Contents after Ewald Breakfast. 

Pathological Variations. 

Table of Diagnostic Characteristics in Certain Diseases. 

Chapter V. 

Feces 77 

Macroscopic Examination. 
Microscopic Examination. 
Chemical Examination. 
Pathological Findings and Significance. 
Parasites and Ova. 

Table of Distinguishing Characteristics of Certain Intes- 
tinal Parasites. 

Chapter VI. 
Human Milk 85 

Chapter VII. 
Cerebrospinal Fluid 87 

Chapter VIII. 
Sputum 93 

Macroscopic Examination. 
Microscopic Examination. 
The Sputum in Disease. 

Chapter IX. 
Bacteriology 97 

Methods of Bacteriological Examinations of Pathological 

Material. 
Culture Media and Making of Cultures. 
Stains and Methods of Staining. 
Diagnostic Characteristics of Pathogenic Bacteria. 
Preparation of Bacterial Vaccines. 

Appendix. 
Forms for Report Blanks 



CHAPTER I. 
BLOOD. 

The routine clinical examination of the blood consists in : 

1. Estimation of the haemoglobin. 

2. Counting the erythrocytes. 

3. Counting the leucocytes. 

4. Examination of the stained film. 

Obtaining the Blood for Examination. — 

A sharp blood lance is needed, one with a spear-shaped 
point being preferable. A Hagedorn needle, such as is used 
for puncturing ear drums, makes an excellent blood lance. 

The front of the lobe of the ear is the best place to obtain 
the blood. The ear is less sensitive than the finger and is 
out of the patient's sight, a good thing in cases of nervous 
children or persons likely to faint at the sight of blood. 
The site of the puncture and the lance are cleansed with 
alcohol. A quick stab is less painful than a punching 
motion. The stab should be deep enough to secure a drop 
of blood without much squeezing. 

Estimation of the Haemoglobin. — 

None of the methods simple enough for common use is 
accurate, but changes in haemoglobin sufficient to have 
clinical significance will be shown by any of them. The 
Tallquist method gives as good results as the Dare or the 
Sahli, except in cases in which the haemoglobin is below 



10 BLOOD 

40 per cent. Below this point it is not easy to match the 
color scale. 

The Tallquist haemoglobin scale consists of a book con- 
taining sheets of absorbent paper and a color scale. A sheet 
of the absorbent paper is touched to a drop of blood large 
enough to make a spot on the paper greater in circumference 
than the round openings in the color scale. When the blood 
has soaked in but not dried, the blood spot is placed behind 
the different openings in the color scale until a match is 
found. In order to secure a white background for the blood 
drop the absorbent paper is folded back of the spot, and the 
color scale with the test paper is pressed flat against the 
cover of the book. 

Theoretically 100 per cent represents the normal amount 
of haemoglobin, but many well persons have haemoglobin 
as low as 80 per cent and not many city dwellers have above 
90 per cent. 

Counting the Corpuscles.- — 

The Thoma-Zeiss Haemocytometer consists of two 
pipettes, one for erythrocytes, one for leucocytes ; and a 
counting chamber, which is formed by a slide with a ruled 
space, and a cover glass. The counting chamber with the 
Turck ruling is preferred. 

Counting Erythrocytes. — 

The red cell pipette has the mark 101 above the bulb. 
For counting erythrocytes a diluting fluid which will pre- 
serve red cells is used. Physiological salt solution may be 
used, but Hayenrs fluid is better. 

Hayem's diluting fluid for erythrocytes: 

Sodium Chloride, 1 gram. 
Sodium Sulphate, 5 grams. 
Mercuric Chloride, 0.5 gram. 
Distilled Water, 200 ex. 



MANUAL OF LABORATORY DIAGNOSIS 11 

fhe pipette is touched to a freshly exuded drop and the 
blood is drawn up to the mark 0.5. Any excess oi blood 
is wiped from the c]\d of the pipette, and the pipette is 
immersed in the diluting fluid, which is drawn up to the 
mark 101. the pipette being rotated the while to mix the 
fluids. Both ends of the pipette are closed with thumb and 
linger and the pipette is shaken until blood and diluting 
fluid are thoroughly mixed. Two or three drops are then 
blown out and a drop is mounted for counting. 

The counting chamber consists of a slide upon which is 
cemented a circular piece of glass, the island. Upon the 
surface of the island one square millimeter is ruled into 400 
small squares. If the counting chamber has the Tiirck 
ruling, this square millimeter is surrounded by eight other 
squares of equal size, with a few intersecting lines in each 
square. 

Surrounding the island and also cemented to the slide is 
a square of glass with a circular hole in the middle large 
enough to encircle the island, leaving a moat or ditch 
between. This square is 0.1 millimeter thicker than the 
island, so that the counting chamber formed when the cover 
glass is placed is 0.1 millimeter dee]). 

When read\- to mount the drop for counting, the end of 
the pipette is wiped, its point brought in contact with the 
island at an angle of 45 degrees and a drop milked out by 
pressing with the Angers on the rubber tubing attached to 
the pipette. The size of the drop can be well controlled in 
this way. The drop must be just large enough to till the 
island and not run over into the moat when the cover glass 
is applied. 

To place the cover glass, take it by one corner and let the 
opposite corner touch the slide at the side of the square, so 
that the cover glass leans against the edge of the square and 
is held in this position by the finger. Steadied in this way, 



12 BLOOD 

it can be settled into place without forming bubbles in the 
counting chamber. 

Allow a few moments for the corpuscles to settle, then 
place the slide under the microscope and take a general view 
of the field with the low-power lens to see if the corpuscles 
are evenly distributed. If they are not even, another mount 
must be made. If they are, the preparation is ready to 
count. A high dry lens is usually used for counting. An 
arrangement of lens and eye piece is desirable which will 
bring sixteen small squares into the field at the same time. 
It will be seen that every fifth small square is intersected 
by a line drawn through the middle, thus, blocks of sixteen 
small squares, none of which have intersecting lines, are 
marked off. 

Count all the corpuscles in five of these blocks of sixteen 
and find their sum. To this sum add four ciphers and the 
result is the number of corpuscles per cubic m.m. 

In counting five blocks of sixteen, eighty small squares 
have been counted, or one-fifth of the whole number of 
small squares (400). Therefore, to find the number of cells 
in the whole square m.m. the number counted is multiplied 
by five. The cubic contents of the counting chamber 
within the ruled square is 0.1 cubic m.m., therefore, multi- 
ply by ten to find the number in a whole cubic m.m. The 
blood has been diluted 200 times in filling the pipette, there- 
fore, multiply by 200 to find the number of corpuscles in 
one cubic m.m. of undiluted blood. 

Suppose the five blocks counted contained 96, 105, 98, 
108 and 95 cells, the whole 80 squares then contain 502 cells ; 
502X5X10X200=5,020,000. The same result is obtained 
by the rule (to the sum of the corpuscles in 80 squares add 
four ciphers). It is customary to disregard figures below 
the hundred-thousand place, so the count in the above ex- 
ample is 5,000,000. 

The normal number is given as 5,000,000, but higher 



MANUAL OF LABORATORY DIAGNOSIS 13 

counts are often found, especially in young - , vigorous adults, 
up to 6,000,000 not being unusual. 

Counting Leucocytes. — 

The leucocyte pipette has the mark 11 above the bulb. 
Acetic acid is used as diluting fluid, as it dissolves the 
erythrocytes and leaves only the leucocytes in the field. 
Diluting fluids, both for erythrocytes and leucocytes, shculd 
be filtered often enough to keep them perfectly clear. 

Diluting fluid for leucocytes: 

Glacial acetic acid, 1 c.c. 
Distilled water, 99 c.c. 

The pipette is filled and the drop mounted, as in counting 
reds. The low power is used for counting leucocytes, as 
with it a square millimeter, which is the unit in counting 
white cells can be got into the field. With a Tiirck count- 
ing slide the central square is ruled into 400 small squares, 
as described in counting red cells, and this square is sur- 
rounded by eight other squares of equal size but less closely 
ruled. These outside squares are better for counting leu- 
cocytes, as there are fewer lines to confuse the eye. A 
good rule is to count all the cells in each corner square, 
average the result and multiply by 200. 

Example. — Suppose the square millimeters counted con- 
tained 45, 50, 47, 42. The average number is 
46. The depth ot the cell is 0.1 millimeter; 
therefore, to find the number in a whole cubic 
millimeter one must multiply by ten. The 
dilution is 20. 45X10X20=9,200, the number 
of cells per cubic millimeter of undiluted blood. 

Cleansing the Haemocytometer. — 

Use nothing on the slide but water. Dry slide and cover 



14 HLOOD 

glass with a soft cloth. Old handkerchiefs should be kept 
for this purpose. 

Blow the fluid from the pipettes, then draw up and blow 
out, first, water, second, alcohol and, third, ether. Lastly, 
draw air through pipette until the glass ball rolls freely. 
A soft rubber bulb, such as is used for an ear syringe, with 
an inch cut off the tube, is useful for cleansing pipettes, 
both to draw up fluids and to blow out fluids and air. 

Color Index. — 

The term color index indicates the haemoglobin content 
of the erythrocyte, as compared with a normal standard. 
Five million is taken as the standard erythrocyte count and 
is called 100 per cent. The standard of haemoglobin is 
100 per cent, as estimated by the ordinary instruments. 
% of haemoglobin. 

Color index = ■ 

' , of erythrocytes. 
Example. — The haemoglobin is 80%. 

The erythrocyte count is 5,000,000 ('100'* •. 
80-^-i00=0.8=c61or index. 

To obtain the per cent of the red cell count, multiply the 
first two figures of the count by two. 

Making and Staining Blood Films. — 

Slides and cover glasses must be clean and well polished. 
Washing in water and drying with a soft cloth is usually 
sufficient. 

Making Films: Touch a slide near its end to a small drop 
of blood. Place one end of a second slide against the sur- 
face of the first and draw it up to the drop of blood so that 
the blood is in the acute angle. Push the spreader slide slowlv 
along the surface of the first slide. The blood will be drawn 
along into a thin film. 



MANUAL OF LABORATORY DIAGNOSIS 15 

Staining Films: Most modern blood stams are made up 
with methyl alcohol, which fixes the film. As soon as the film 
is dried in the air it is ready for staining. 

Wright's Blood Stain is the most satis factor)- for general 
use. It is difficult to prepare and can be satisfactorily pur- 
chased. That obtained from E. II. Sargent & Company is 
reliable. The original technique for staining gives the best 
results. 

1. Drop stain upon air dried film until the film is well cov- 

ered. Allow to stand one minute. 

2. Drop on distilled water until metallic lustre appears. It 

usually takes a little more water than stain. Allow to 
stand three or four minutes. 

3. Wash in distilled water until film looks pink. Drain or 

dry with filter paper. Examine with oil immersion lens. 

Effect of the Stain. 

1. Erythrocytes stain pink or yellowish pink. 

2. All nuclei stain blue or purplish ; nuclei of lymphocytes 

deep purplish blue, of large mononuclear leucocytes 
lighter blue, of granular cells dark blue. 

3. Cytoplasm of lymphocytes stains robin's ^gg blue, of 

large mononuclears transparent pale blue, of granular 
cells faintly pink or not at all. 

4. Granules. Neutrophile granules stain violet, eosino- 

phile granules bright red, basophile granules dark 
purplish blue. 

5. Rlood platelets stain blue or lilac. 

6. Malarial parasites stain blue, with the chromatin body red- 

dish purple. 

7. liacteria stain blue. 



16 BLOOD 

Leucocytes, varieties, percentage of each kind and appear- 
ance in the stained film. 

1. Small lymphocytes. 15% to 30%. Smaller than granular 

leucocytes. (7 to 11 /*.) The nucleus is large, round, 
deeply staining and is surrounded by a relatively small 
amount of cytoplasm which takes the basic stain 
(light blue). 

2. Large mononuclear cells. 3% to 10%. As large as or 

larger than granular cells, 
a. Large lymphocytes. Differ from small lymphocytes only 
in size or sometimes in staining less deeply. Not 
numerous in adult normal blood. 

b. Large mononuclear leucocytes. The nucleus is rela- 
tively small, single, round or oval, and is surrounded 
with a wide zone of faintly staining cytoplasm. 

c. Transitional cells. Differ from large mononuclear leu- 

cocytes only in having a deeply indented or horseshoe- 
shaped nucleus, 
a, b and c are usually classed together in making a differ- 
ential leucocyte count. 

3. Polymorphonuclear neutrophils. 60% to 75%. Average 

size 11 w. Irregular convoluted nucleus, often looking 
like separate nuclei. Cytoplasm filled with fine dust- 
like granules stained lilac color or dull pink. 

4. Eosinophils. 1% to 4%. Differ from neutrophiles only 

in having coarse round granules which stain bright red. 

5. Basophiles or mast cells. 0.5% to 1%. Differ from neu- 

trophiles in having large irregular-shaped granules which 
stain a deep purplish blue. 
3, 4 and 5 are the granular leucocytes. The granular leu- 
cocytes, the large mononuclear leucocytes and transition- 
als are formed in the bone marrow*; the large and small 
leucocytes in lymphatic tissue. 



MANUAL OF LABORATORY DIAGNOSIS 17 

The Differential Leucocyte Count. 

The differential leucocyte count is made from the stained 
film, using the oil immersion lens. A list is made of the .varie- 
ties of leucocytes, polymorphonuclear neutrophiles, small 
mononuclears, large mononuclears (including large lympho- 
cytes, large mononuclear leucocytes and transitionals), eosino- 
philes, basophiles and myelocytes. The slide is examined 
systematically, so as to avoid counting the same area more 
than once, and each leucocyte seen is recorded under the proper 
heading. One hundred leucocytes are counted in this way. 
The number recorded under each heading is the percentage of 
that variety. Except when the first hundred is quite normal, 
the total leucocyte count being normal also, another hundred 
should be counted and the results averaged. When the differ- 
ential count is of special importance, at least four hundreds 
should be counted. 

Beginners usually set down a stroke for each cell counted, 
making four and crossing with the fifth. With practice a 
considerable number can be kept in the mind, so that it is 
necessary to record only a few times during- the count, 
using figures instead of strokes. 

Coagulation Time. 

As a preliminary to surgical operations, as tonsillectomy or 
any surgical procedure in the presence of jaundice, it is 
often important to test the coagulability of the blood. The 
coagulation time is much increased in haemophilia, purpura 
and jaundice. 

A modification of Rudolph's method is practical. A wide- 
mouthed bottle containing about one litre is fitted with a large 
cork, which is perforated for several glass tubes and a ther- 
mometer. The glass tubes may be made from tubing about 
one-fourth inch in diameter. They are long enough to reach 
nearly to the bottom of the bottle and extend a little above the 
cork, and are sealed at the lower end. The bottle is filled with 



18 BLOOD 

water, at the temperature of 20" C. Several capillary pipettes 
about one-sixteenth inch in diameter and as long as the tubes 
are each fitted with a rubber nipple. The ear or finger is 
punctured and the time of appearance of the drop noted. 
Enough blood is drawn into one of the capillary pipettes so 
that the column is two inches or more in length. It is then 
drawn high enough so that it is an inch or more from the end 
at which it entered. This end is sealed in the flame and the 
pipette inserted into one of the tubes. It is well to fill two or 
three pipettes in this manner. The blood of the person mak- 
ing the test may be used as a control if it is known to be 
normal . 

At the end of four or five minutes from the time the punc- 
ture was made, draw out the first of the pipettes, score with 
a sharp file near the lower end of the column, break the pipette 
and separate the ends slowly. When the blood has coagulated 
the clot will draw out between the broken ends. If coagula- 
tion has not taken place other trials are made at intervals of a 
half minute until the clot is demonstrated. Normal blood 
coagulates in from five to eight minutes, as a rule. If the 
coagulation time is more than ten minutes it is distinctly 
retarded. 

General Pathology of the Blood. 

Erythrocytes may vary from the normal : 

In number. — 

1- Oligocythaemia is a decreased number of erythrocytes. 

Tt occurs in : 

a. All anaemias. 

1). Blood dilution. This is a temporary condition after 
administration of salt solution or when transudates 
are being absorbed. 



MANUAL OF LABORATORY DIAGNOSIS 19 

2. Polycythaemia is an increased number of erythrocytes. 
This is not a frequent finding. 

It occurs in: 

a. Loss of fluids, as after excessive diarrhoea, sweats or 

vomiting. 

1). High altitudes, proportional to the height, 

c. Phosphorus and CO poisoning. 

(1. Cyanosis, from any cause. 

e. The new-born, 

f. A few cases of very high counts of unknown origin. 

In size. — 

Macrocytes are erythrocytes above the normal size. 
Microcytes are erythrocytes below the normal size. 
The presence of macrocytes and microcytes is called ani- 
socytosis. 

In shape. — 

The presence of red cells abnormal in shape is called poi- 
kilocytosis. 

In staining properties.— 

Polychromatophilia is a condition in which the red cells take 
other than the acid stain ( eosin ) : that is. they stain more 
or less with the basic dye (methylene blue) and appear 
bluish or lead colored instead of pink. Basic stippling is 
a condition in which the basic -tain shows in fine specks 
or dots. 

In being nucleated. — 

Normoblasts are nucleated reds of normal size. 
Megaloblasts are nucleated reds of larger than normal size. 
Aficroblasts are nucleated reds of smaller than normal size. 



20 BLOOD 

Megaloblasts are usually young cells with large, pale, reticu- 
lated nuclei and cytoplasm containing but little haemo- 
globin and therefore staining a bluish color. Normo- 
blasts are usually older cells with smaller, deeply stain- 
ing nuclei, and haemoglobin containing cytoplasm staining 
like non-nucleated reds. Microblasts are old, degenerat- 
ed cells having often but little cytoplasm left and that 
taking the basic stain. Their nuclei stain very deeply. 
The presence of nucleated reds is abnormal in the circu- 
lating blood (except normoblasts in the new-born) and is 
evidence of abnormal stimulation of the blood forming 
marrow. The appearance of megaloblasts is of more 
serious significance than the appearance of normoblasts. 

Leucocytes. — 

Leucocytes vary from the normal in number, in propor- 
tion of the different varieties, and in the appearance of the 
myelocyte, normally found only in the bone marrow. 

The myelocyte is a large mononuclear cell with gran- 
ules. The nucleus is round, oval or slightly indented and 
often eccentrically placed. There are three varieties, named 
according to their granules, neutrophilic myelocytes, eosi- 
nophilic myelocytes and basophilic myelocytes. They stain 
less perfectly than normal leucocytes, both as to nuclei and 
granules, and most of them are larger (18 /x or 20 /*). These 
cells are the parent cells of the granular leucocytes. 
Myeloid leukemia is the only condition in which myelo- 
cytes appear in the circulating blood in large numbers. A 
few may be present in any severe anaemia or pronounced 
leucocytosis. 

Leucocytosis is an increase in the number of leucocytes in 
the circulating blood. The term is commonly used to 
mean that kind of leucocyte increase which appears as a 
response to most infections, that is, a polynucleosis. 



MANUAL OF LABORATORY DIAGNOSIS 21 

Leucocytosis (polynucleosis) occurs: 

1. Physiologically. 

a. In the new-born, lasting a few days. 

b. After a full meal (not pronounced). 

2. Pathologically. 

a. In most infections and febrile diseases, except typhoid, 

malaria, measles, influenza and uncomplicated tuber- 
culosis. 

b. After hemorrhage. 

c. In malignant disease. 

d. From various toxic and medicinal causes. 

Lymphocytosis is an increase in lymphocytes, either abso- 
lute (increase in total number) or relative (increase in 
percentage). 

It occurs : 

1. Physiologically, in childhood (relative). 

2. Pathologically. 

a. In whooping cough (absolute as w^ell as relative), may 

be high and may occur early enough to aid in diag- 
nosis. 

b. In syphilis, especially hereditary (relative). 

c. In malaria (relative). The large mononuclear is in- 

creased. 

d. In leucopenia from any cause (relative), due to de- 

crease of polynuclear cells, for example in typhoid 
and pernicious anaemia. 

e. In lymphatic leukemia. The highest total count and 

greatest proportion of lymphocytes occur in this 
disease. 

« 
Eosinophilia is an increase in eosinophiles in the circulat- 
ing blood. 



11 BLOOD 

1 1 occurs : 

1. In bronchial asthma. 

2. In skin eruptions from any cause. 

3. When there are intestinal parasites, notably in trichi- 

nosis. 

4. In myelogenous leukemia. Eosinophils are increased 

with every form of granular cells. 

Increase in Basophiles or Mast cells. — 

1. In myeloid leukemia. 

2. AYhenever eosinophils are increased, basophiles are apt 

to be somewhat increased. 



Blood Diseases. 

Anaemia is a deficiency in red cell substance, that is, in 
haemoglobin, in number of red cells or in both. 

Secondary anaemia. — 

Typical blood picture : — 

Haemoglobin reduced. 

R. B. C. reduced. 

Color index low. 

\Y. B. C. moderate leucocytosis. 

Stained him: qualitative changes only in severe cases. 

Normoblasts, rarely megaloblasts. may be present. 

Chief causes, and special characteristics. 

1. Acute hemorrhage, as from trauma, abortion, tubal preg- 
nancy, typhoid, pulmonary tuberculosis. 



MANUAL oi : LABORATORY DIAGNOSIS 23 

anaemia appears quickly and recovers quickly, the 
count becoming normal sooner than the haemoglobin. 

2. Chronic hemorrhages, as from hemorrhoids, fibroid tu- 

mors of the uterus, cancer of the stomach. 
Haemoglobin may be very low, the red cell count not so 
low. fewer blasts, recover}- slow. 

3. Blood poisons. 

a. Infectious diseases. 

b. Malignant tumors, the degree of anaemia in proportion 

to malignancy. 

c. Chemical poisons, e. g., lead, mercury, arsenic and the 

coal tar derivatives. In lead poisoning basic stip- 
pling is found early and blasts are numerous. 

d. Chronic wasting diseases, e. g., lues, nephritis, rickets. 

4. I'oor food, inanition and unhygienic surroundings. 

5. Parasites, as .hookworm, sometimes tapeworm. The 

anaemia may be severe and show qualitative changes 
in red cells. 

6. Malaria. Anaemia results from direct destruction of red 

cells. There is a relative increase of the large mono- 
nuclear, but no increase in the total leucocyte count. 
There may be leucopenia. Late severe malarial 
cachexias show a blood picture like pernicious 
anaemia. 

Chlorosis. — 

Chlorosis is a disease of defective blood formation. There 
is no evidence of blood destruction. It is not diagnosed by 
tlie blood alone. 

Blood picture : — 

Haemoglobin low. This is the essential change. R. P>. C. 
count moderately low. or may be normal. Color index verv 



24 BLOOD 

low, often 0.5. W. B. C. count normal or slightly reduced. 
Stained film: All the red cells show lack of haemoglobin 
and are slightly undersized. Qualitative changes are absent 
or inconspicuous. Normoblasts may be found in severe 
cases. Blood plates are increased. There is a tendency to 
relative lymphocytosis. 

Pernicious Anaemia. 

The etiology is unknown. Haemolysis is the chief change. 
Whether the haemolytic agent acts in the blood-making 
marrow or in the circulation is not known. The only lesions 
constantly found post-mortem are hyperplasia of red bone 
marrow and increased iron pigment in the liver. The dis- 
ease is nearly always finally fatal, but three or four remis- 
sions may occur, lasting from a few days to many months. 
The blood examination is necessary to a diagnosis, but clin- 
ical history and symptoms are also important. 

Blood picture : — 

Appearance, pale, watery and slow to coagulate. 

Haemoglobin, low but not so reduced as the red cell 
count- R. B. C. count very low, the most striking change, 
(1,000,000 to 2,000,000 common, has been reported as low 
as 150,000). 

Color index high, the only condition in which it is high. 
The higher the color index the worse the prognosis. 

W. B. C. count low. Leucopenia may be pronounced 
(3,500 common, has been reported as low as 500). 

Stained smear: Cells scattered in the field. Qualitative 
changes marked, poikiloc} r tosis, anisocytosis (more macro- 
cytes than microcytes), polychromatophilia and basic stip- 
pling all present in typical cases. Variation in haemoglobin 
content is shown by dark and pale staining cells. The 



MANUAL OF LABORATORY DIAGNOSIS 25 

more macrocytes with excess of haemoglobin, the higher the 
color index. Nucleated reds are present in all cases, but 
vary in number at different times. Typically, megaloblasts 
exceed normoblasts. The differential leucocyte count shows 
a relative lymphocytosis proportional to the leucopenia. 
Eosinophils are decreased when the disease is progressing 
.and increased when there is improvement. An occasional 
myelocyte may be found. Blood plates are reduced in 
number. 

Leukemia. — 

Leukemia is a condition characterized by persistent in- 
crease in leucocytes, and changes in lymph glands, spleen 
and marrow. 

There are two types of blood findings: 1st, the myeloid, 
in which there is an overproduction of those leucocytes 
formed in the marrow, that is, all kinds except lymphocytes; 
2nd, the lymphatic, in which there is an overproduction of 
lymphocytes. 

In the myeloid form there is hyperplasia of the myeloid 
marrow, and myeloid tissue may appear in the spleen, 
lymph glands, liver and many other places in the body. In 
the lymphatic form lymph tissue may infiltrate the various 
organs and tissues of the body. Some pathologists regard 
leukemias as malignant tumors of the marrow and of lym- 
phatic tissue. The tumor cells, being- cells belonging to the 
circulating blood, are carried to all parts of the body and 
form metastases. 

Either form of the disease may be acute or chronic in its' 
course. Remissions sometimes occur in which the leucocyte 
count may be normal, but examination of a stained smear 
usually shows the presence of abnormal cells and abnormal 
percentages of the different varieties of leucocytes. 
Blood picture in myeloid leukemia. (Synonyms, myelogenous 
leukemia, splenomyelogenous leukemia, myeloblastoma.) 



26 BLOOD 

Haemoglobin is reduced as the disease progresses. Esti- 
mation by usual methods is difficult because the large num- 
ber of leucocytes changes the color and consistency of the 
blood. 

. R. B. C. count is little reduced at first, more later. 
Color index low. 

W. B. C. count enormously increased (250,000 common, 
may be 1,000,000). 

Stained film : The diagnosis is often made at a glance 
from the great number of granular leucocytes in the field 
The differential count shows many myelocytes (30% aver- 
age). All kinds of leucocytes except lymphocytes are in- 
creased. The percentage of the different varieties varies 
greatly. Qualitative changes in the reds are usual. Blasts 
are constantly found and are more numerous than in any 
other blood disease. 

Blood picture in lymphatic leukemia. (Synonyms, lymphaemia, 
lymphoblastoma. ) 

Haemoglobin low. 

R. B. C. count low. 

Color index low. 

W. B. C. count very high (150,000 common, may be 
1,000,000, but is often as low as 30,000). Some acute cases 
show very moderate counts. 

Stained film : Although the erythrocyte count is often 
lower than in myeloid leukemia on account of hemorrhages 
which are frequent in this condition, the qualitative 
changes are not so marked and nucleated reds are much 
fewer. The differential count usually shows 90% or more 
of lymphocytes. The other varieties of leucocytes are not 
much changed. 



MANUAL OF LABORATORY DIAGNOSIS 11 

Diagnosis of Malaria from the blood examination. - 

The plasmodia of malaria arc found in the circulating 
blood during and shortly before and after febrile paroxysms. 
Their presence during quiescent periods is not easy to 
demonstrate. Quinine, even one dose, causes most of the 
parasites to disappear from the peripheral blood. 

Methods of Examination : — , 

1. Moist preparation. Take a small drop of blood on a 
slide, cover with cover glass, pressing upon the cover glass 
to make the layer of blood thin. Examine at once. 
The parasites appear as hyaline, variously shaped bodies in 
the red cells. Except in their earliest stages they contain 
dark, brownish pigment which has a dancing motion. The 
pigment is usually the first thing to attract the eye. The 
parasite itself, except in the full-grown asexual and sexual 
forms, has a slow amoeboid motion. 

2- The stained film. This method is preferred when the 
patient is not convenient to the laboratory. It is less liable 
to error in the hands of the inexperienced. 

With Wright's stain the parasite stains blue with a red- 
dish chromatin body within it. The pigment is unchanged, 
looking about the same as in the unstained blood. 

In tertian and quartan fevers the asexual forms are numer- 
ous in the peripheral blood and the occasionally found sex- 
ual forms are not easily distinguished from the full-grown 
asexual. 

In aestivo-aiitinnnal fever the asexual forms are at first 
seen in the peripheral blood, but their later development takes 
place in the spleen. The sexual form of this variety, the 
crescent, is distinctive and is constantly found in the periph- 
eral blood after the first few days of the infection. These 



28 BLOOD 

crescents are resistant to quinine and often remain in the blood 
a long time. 

The blood in malaria early in the infection may show noth- 
ing abnormal but the presence of the parasites. Anaemia 
develops rapidly and in long-standing cases the blood may 
show a picture much like that of pernicious anaemia. 



CHAPTER II. 

SERUM DIAGNOSIS. 

Widal Reaction. 

The Typhoid Culture.— 

Old laboratory cultures which have been many times 
transplanted are often better, being more motile than fresh- 
ly-isolated bacteria. For making the test use an 18 to 24- 
hour bouillon culture which has been inoculated from the 
stock agar culture and incubated. At this stage the bacilli 
are actively motile and are about numerous enough to make 
a good field. Examine a loopful of the culture under the 
high dry lens to see that the bacilli are motile and are not 
spontaneously clumped. 

Obtaining the Blood or Serum. — 

The blood is obtained from a puncture as for a blood 
count. AVhole blood may be used. It can be diluted in a 
white blood counting pipette. The disadvantage is that 
the corpuscles obscure the field. Blood serum is the best 
form to use. Obtain the blood in a capillary tube (Wright 
capsule), centrifuge to separate the serum, or let stand until 
the serum separates. Dried blood may be used, will keep 
for some time and is convenient for sending to the labora- 
tory. Large drops of blood are allowed to drop on glass 
slides or glazed paper and are dried in the air. 

29 



30 SERUM DIAGNOSIS 

Dilution of the Serum. — 

A dilution of 1 to 40 or higher is required that the test 
may have any diagnostic value, because normal blood undi- 
luted or 1 to 20 will sometimes clump typhoid bacilli. 

For whole blood. — Draw the blood to 1 mark in white 
blood counter, draw up physiological salt solution to mark 
11 and mix. This, makes 1 in 20 dilution (blood is half 
serum). Blow out into a watch glass. 

For serum. — Remove the serum with capillary pipette- 
To one drop of serum add 19 drops of physiological salt 
solution. This makes 1 in 20 dilution. 

For dried blood. — Scrape the dried blood from the slide 
into a watch crystal. Add 9 drops of salt solution. This 
makes dilution 1 in 20 (dried blood is half serum). 

Making the Mixture. — 

Place a loopful of the 1 in 20 dilution of serum on a cover 
glass. Mix with a loopful of the bouillon culture. The 
fluid of the culture doubles the dilution, making 1 in 40 
dilution of the serum. Ring a hollow ground slide with 
vaseline and mount the mixture as a hanging drop. 

Observing the Reaction. — 

Use the high dry lens and subdued light. Artificial light 
is sometimes better than daylight. The field should show 
at first actively motile separate bacilli. If the reaction is 
positive, in from a few minutes to 1 hour the motion gradu- 
ally slows and ceases, and the bacilli gather into groups. 
The reaction is more or less marked according to the 
amount of agglutinin present. To call a reaction positive 
the majority of the bacilli must have stopped their motion 



MANUAL OF LABORATORY DIAGNOSIS 31 

and there must be many clumps of 5 or more bacilli. For 
a 1 in 40 dilution 40 minutes' time of observance is enough ; 
for higher dilutions. 1 hour. 

Value of Widal Reaction in Typhoid Fever. 

In typhoid fever the Widal reaction rarely fails to appear, 
and is our most certain confirmatory test after the bacilli 
have left the blood. During the first week of the disease 
the typhoid bacilli may be found in the blood. The Widal 
appears first on the seventh or eighth day. usually continues 
well into convalescence, and may continue an indefinite 
time after recovery. 

The Wassermann Test for Syphilis. 

Haemolysis. — 

If the erythrocytes of an animal, for example, a sheep, 
are injected into the blood stream of an animal of another 
species, for example, a rabbit, the blood of the animal receiv- 
ing the corpuscles (the rabbit ) gains power to dissolve the 
erythrocytes of the species from which the corpuscles come 
(sheep) that is. becomes immunized to these corpuscles. 
But if the serum of the rabbit is heated to 56" C. for one-half 
hour (inactivated), or kept at room temperature for twenty- 
four hours, it loses this power. Haemolysing power may 
be restored to this serum by adding to it some fresh un- 
heated serum from any animal. The haemolysis depends 
upon two substances present in the serum of the immunized 
animal, one destroyed by heat (complement) and the other 
not so destroyed (amboceptor). 

A haemolytic system consists of a suspension of erythro- 
cytes, the serum of an animal immunized to these erythro- 
cytes, and complement (contained in fresh unheated serum 
of any animal). 



32 SERUM DIAGNOSIS 

Complement Fixation. — 

Complement is present in all sera and will react with 
different kinds of amboceptors, but amboceptors are devel- 
oped as the result of the introduction into the blood of some 
foreign material ; for example, erythrocytes of another spe- 
cies give rise to haemolytic amboceptors. Bacteria give 
rise to bacteriolytic amboceptors, which react in a similar 
manner with complement and the invading bacteria. 

The substance giving rise to these amboceptors is called 
antigen ; the corpuscles are the antigen in the haemolytic 
system, the bacteria are the antigen in the bacteriolytic 
system. 

If a bacteriolytic antigen, the corresponding amboceptor 
and complement are incubated together until the combina- 
tion has had time to take place, and then the antigen of a 
haemolytic system, that is, erythrocytes, and haemolytic 
amboceptor are added to the former combination, no haemol- 
ysis will take place, because the complement has been used 
up or bound in the first system. If, however, the first sys- 
tem contained no amboceptor, then haemolysis would take 
place, as the complement would be free to unite with the 
haemolytic system. 

The Wassermann reaction in syphilis seeks to demon- 
strate, by this method, the presence or absence of syphilitic 
amboceptor in a patient's blood. 

In general terms the proceeding in the Wassermann reac- 
tion is this : The patient's blood serum, inactivated to 
remove its own complement, is combined with complement 
supplied by fresh guinea pig serum, and syphilitic antigen. 
These three substances are incubated together until com- 
bination of complement, antigen and amboceptor has had 
time to take place if the patient's serum contains syphilitic 
amboceptor. There is no visible change to show whether 



MANUAL OF LABORATORY DIAGXOSIS 33 

combination has taken place or not. If the patient's serum 
contains syphilitic amboceptor and this has combined with 
the antigen and complement, then there will be no free 
complement left in the mixture. Adding another system 
of antigen and amboceptor, one in which the combination, 
if it takes place, will cause a visible change, will then show 
whether there is complement in the mixture free to act in 
the latter system. A haemolytic system is used, as haemol- 
ysis is a plainly visible phenomenon. 

After an hour's incubation haemolytic amboceptor (the 
serum of a rabbit immunized to sheep's erythrocytes) and 
washed sheep's corpuscles are added and the mixture again 
incubated. If there is free complement it will unite with 
the haemolytic amboceptor and sheep's corpuscles and dis- 
solve the corpuscles. If the complement has been used in 
the former combination, the sheep's cells will not be dis- 
solved. Absence of haemolysis, then, indicates that the 
patient's blood contains syphilitic amboceptor, that is, the 
reaction is positive. Haemolysis shows that the comple- 
ment was not used in the first combination, owing to the 
absence of the third necessary element, syphilitic ambocep- 
tor in the patient's blood, but was free to enter into the 
haemolytic system and bring about haemolysis of the 
sheep's cells, that is, a negative reaction. 

Preparation of Materials Used in the Test. — 

(1) Sheep Corpuscles. — If the sheep's blood is obtained 
from a slaughter house it must be used within a day or two, 
as bacterial contamination will soon cause haemolysis. 
But if obtained in a sterile manner and kept sterile the 
corpuscles may be used for a week, as a rule. In either 
case, as soon as the blood is drawn it must be defibrinated 
by shaking five minutes in a bottle with bits of glass or 
wire. It is then kept in a refrigerator until time to prepare 
for use. 



34 SERUM DIAGNOSIS 

(2) Haemolytic Amboceptor. — The inactivated serum of 
a rabbit which has been immunized to sheep erythrocytes 
furnishes the haemolytic amboceptor. Two intravenous 
injections of one to two c.c. of washed sheep corpuscles are 
given to a rabbit at an interval of four or live days. Five to 
seven days after the second injection the rabbit's serum will 
contain the maximum amboceptor. At this time the rabbit 
is bled from the ear vein, the serum tested, and if found of 
sufficient strength to use in a dilution as high as 1 to 1,000, 
the rabbit is anaesthetized and bled from the carotid with 
aseptic precautions. The serum is separated by the centri- 
fuge and inactivated at 56° C. for one-half hour, to destroy 
its complement. It is well to inject two or three rabbits 
at a time, as not every one injected produces good ambo- 
ceptor. The amboceptor is stored in sealed glass tubes 
and kept in the refrigerator. 

(3) Complement. — Guinea pig serum is used as its com- 
plement content is high and rather constant. The animal 
may be bled by puncturing the heart with a hypodermic 
needle and withdrawing four or live c.c. of blood into the 
syringe. This requires some skill and many prefer to kill 
the pig and bleed from the vessels of the neck. The blood 
should stand until coagulation has taken place. The serum 
is separated by the centrifuge and pipetted from the clot. 
It must be used within twenty-four hours unless it is kept 
frozen. The serum can be placed in small tubes with water- 
tight stoppers and buried in salt and ice in a thermos bot- 
tle. By renewing the salt and ice every day or two the 
complement will be kept frozen and will remain potent for 
weeks. lust what is needed for a set of tests may be melted 
at the time the tests are made. 

(4) Antigen. — The substances used as antigens in the 
Wassermann reaction are not biologically specific antigens. 
Various lipoid substances have been found to bind comple- 



MANUAL OF LABORATORY DIAGNOSIS 35 

ment in the presence of syphilitic serum and not to hind it 
in the presence of non-syphilitic serum. 

Among the substances found most successful as antigens 
are, first, alcoholic extracts of syphilitic liver ; second, alco- 
holic extracts of normal organs, as guinea pig hearts ; third, 
cholesterinized extracts of normal organs, as guinea pig, 
beef or human heart muscle. These extracts are not always 
of the same strength nor enduring quality. 

(5) The Patient's Blood Serum. — The blood is obtained 
from a vein at the bend of the elbow. A ten c.c. Luer 
syringe with a No. 19 needle is convenient for puncturing 
the vein and obtaining the blood, or the blood may be 
allowed to drop directly through the needle into a sterile 
tube. It is well to take as much as 5 c.c. of blood, although 
less may be sufficient. The blood is allowed to stand until 
coagulated. The serum is separated by the centrifuge and 
pipetted from the clot. The serum is inactivated in a water 
bath at 56° C. for one-half hour to destroy its complement. 
If the serum is not used within a few hours it should be 
kept in a refrigerator. If handled in a manner to prevent 
contamination the serum will be good for the test for a few 
days, but more reliable results are obtained if the test is 
made within forty-eight hours after the blood is taken. 

The glassware should be clean and dry. All tubes and 
pipettes should be thoroughly washed in hot running water. 
A supply of test tubes ^x4 inches is required. This size 
allows the contents to be well mixed by shaking. 

Racks to tit the tubes with holes arranged in parallel 
rows are useful- Five rows of a dozen in a row form a good 
rack. A water bath with the same arrangement for tubes is 
desirable. One c.c. outflow pipettes marked in tenths are 
used and a good supply of them is needed. 

Sterile centrifuge tubes, and a good supply of capillary 
pipettes with rubber nipples, should be on hand. 



36 SERUM DIAGNOSIS 

0.85% sodium chloride solution is used for diluting all 
reagents. 

Diluting and Titrating Reagents Ready for Test. 

1. Corpuscles. — A few c.c. of the defibrinated sheep's 
blood are put into a centrifuge tube, the tube filled with 
0.85% sodium chloride solution and centrifuged until the 
corpuscles are settled. The fluid is pipetted off, and the 
washing repeated twice, mixing the corpuscles well with the 
salt solution each time. After the last washing all the salt 
solution is carefully pipetted off. A 2.5% suspension of the 
sheep cells is made by taking 1 c.c. of the cells and 39 c.c. 
of salt solution ; 0.5 c.c. of this suspension is used in the test. 

2. Amboceptor. — The amboceptor may be diluted 1-1000 
for the first trial. In order not to waste amboceptor it is 
well to dilute one drop with nine drops of salt solution, and 
make the higher dilutions from this. 0.1 c.c. of this 1-10 
dilution added to 9.9 c.c. salt solution will make the 1-1000 
dilution. 

To titrate the amboceptor when the strength of the com- 
plement is still unknown it is necessary to use a probable 
excess of complement; 0.5 c.c. of a 1-10 dilution of comple- 
ment will usually be enough. 

A series of tubes is prepared, each containing 1 c.c. salt 
solution, 0.5 c.c. corpuscle suspension, and 0.5 c.c. comple- 
ment dilution 1-10. To these add increasing doses of 
1-1,000 dilution of amboceptor, to the first 0.1 c.c, to the 
second 0.2 c.c, and so on. Incubate one-half hour. The 
first tube which shows complete haemolysis contains one 
unit of amboceptor. Two units are used in the test. 

3. Complement. — The amount of guinea pig serum 
needed for the set of tests is diluted 1-10 with salt solution. 
To titrate the complement a series of tubes is prepared, 



MANUAL OF LABORATORY DIAGNOSIS 37 

each containing 1 c.c. salt solution, 0.5 c.c. corpuscle sus- 
pension and one unit of amboceptor. To these are added 
increasing amounts of complement, to the first 0.1 c.c, to 
the second 0.2 c.c, and so on. The tubes are incubated 
one-half hour. The first tube which shows complete hae- 
molysis contains one unit of complement. One and one- 
half or two units are used in the test- 

4. Antigen. — Antigen must be titrated to obtain its anti- 
complementary unit and also its antigenic unit. When 
these units are once obtained they usually remain the same 
for some time, and these titrations need not be repeated 
every time the test is made. 

The anticomplementary unit. — 

Antigens alone absorb some complement, and if used 
in large enough amount would prevent haemolysis in a 
haemolytic system without the presence of any serum or in 
the presence of a normal serum. The amount of antigen 
which will absorb or bind the two units of complement used 
in the test is the anticomplementary unit. To ascer- 
tain this a series of tubes is prepared, each containing 1 c.c. 
of salt solution, two units of complement, 0.1 c.c. of known 
normal serum- To these increasing amounts of antigen are 
added. The first tube may have 0.1 c.c. of a 1-10 dilution of 
antigen, the second 0.3 c.c, etc- These are incubated one- 
half hour in a water bath, then to each is added two units of 
amboceptor and 0.5 c.c. of corpuscle suspension. Incubate 
again for one-half hour- The first tube which shows im- 
perfect haemolysis contains the anticomplementary unit of 
antigen. Not more than one-fourth of this amount should 
be used in the test. 

The antigenic unit. — 

This is the amount of antigen necessary to inhibit haemol- 
ysis with a syphilitic serum. To obtain this unit a series 
of tubes is prepared, each containing 1 c.c salt solution, two 



3$ SERUM DIAGNOSIS 

units of complement and 0.1 c.c. of known syphilitic, serum. 
To these are added decreasing amounts of antigen, begin- 
ning with J /\ the anticomplementary unit. These are incu- 
bated one-half hour in a water bath and then to each tube 
two units of amboceptor and 0.5 c.c. of corpuscle suspen- 
sion are added. The tubes are again incubated for one 
hour. The first tube which shows inhibition of haemolysis 
contains the antigenic unit. This should be much less than 
one-fourth the anticomplementary unit in a good antigen. 
An abundance of antigen should be used in the test. One- 
fourth the anticomplementary unit is often used rather than 
smaller amounts. A fresh dilution of the antigen is pre- 
pared for each set of tests, according to the strength of the 
antigen. It is convenient to dilute the antigen enough so 
that the amount used in the test is contained in 0.5 c.c. of 
the dilution. 

The Patient's and Control Sera. 0.1 c.c. of inactivated 
serum is used in the tests. 

Making the Test. 

These titrated and diluted reagents and the inactivated 
sera are arranged in convenient order. Tubes ^x4 inches 
are numbered in blue pencil as indicated in the chart. The 
chart indicates the order of the tubes in the rack and the 
contents of each tube. The rows are lengthened for other 
sera to be tested and other controls. More rows are added 
for other antigens, if more than one is used. At the end 
of two hours the reaction is usually complete, but it is well 
to place the tubes in the refrigerator and make a final read- 
ing next morning. The front row of tubes, 1, 3 and 5, con- 
taining no antigen' should show complete haemolysis. 
The second row, 2, 4 and 6, will show no haemolysis in 2, 
haemolysis in 4, and haemolysis in 6 if the unknown serum 
is negative and none or little of it is positive. 



MANUAL OF LABOR* i ORY DIAGNOSIS 



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40 SERUM DIAGNOSIS 

The test as described provides for the use of one anti- 
gen, but it is desirable to use two or more as in this way 
the tests are repeated. Sera which contain small amounts 
of antibody may give a clearer positive with one antigen 
than with another. 

The antibody in a positive serum may be titrated by 
using decreasing amounts of the serum. A strongly posi-' 
tive serum will give a positive reaction with a fraction of 
the standard amount used in the test. 

By substituting drops for tenths c.c. throughout, the Was- 
sermann reaction may be made with smaller amounts of 
material. While this method is less accurate, it can be 
used with good results and when the serum is too small 
in amount for the c.c. method, it proves very convenient. 

The Wassermann Test on spinal fluid is made just as the 
test on blood serum is made except that the spinal fluid is 
not inactivated, and that five to ten times the amount is 
used, that is 0.5 c.c. to 1 c.c. of undiluted fluid. 

Diagnostic Value of the Wassermann Test. 

A positive Wassermann may be obtained as early as four 
or five weeks after infection with syphilis, that is, a few 
days after the appearance of the chancre, but as a rule the 
reaction first becomes positive seven or eight weeks after 
infection and remains so throughout all stages of the dis- 
ease if the case is untreated. In hereditary syphilis the 
reaction is positive in an equally high percentage of cases. 
There are occasional exceptions to this rule, a negative 
Wassermann being found in undoubted cases of active 
syphilis. Treatment renders the reaction negative in many 
cases, but a positive reaction recurs if the treatment has 
been insufficient to cure the disease. Three weeks should 
elapse after the cessation of treatment before the test is 
made. In syphilitic diseases of the nervous system the 
blood Wassermann may be negative and the spinal fluid 
Wassermann positive. 



MANUAL OF LABORATORY DIAGNOSIS 41 

Positive Wassermann reactions have been found in some 
cases of jaundice, leprosy, relapsing fever, frambesia, 
trypanosomiasis and malaria in the febrile stage. The in- 
gestion of alcohol may change a positive reaction to a 
negative. 

Complement Fixation Test for Gonorrhoea. 

The complement fixation test for gonorrhoea is per- 
formed in the same manner as the Wassermann test for 
syphilis, substituting gonococcus antigen for syphilitic 
antigen. Gonococcus antigen made from many strains of 
gonococci can be purchased from Parke, Davis & Co. 

The diagnostic value of the complement fixation test for 
gonorrhoea is considerable, a positive reaction being more 
valuable than a negative. The test is seldom positive dur- 
ing the first few weeks of the infection but later in the 
course of the disease a large percentage of the cases show 
positive reactions. 



CHAPTER III. 

URINE. 

Obtaining Specimens. 

A twenty-four hour specimen is usually desirable, be- 
cause the total output of both solids and water is learned 
in this way. A single specimen is sometimes useful, as 
sugar or albumin may be present in the urine at one time 
of the day, and not at other times. 

Under ordinary circumstances no preservative is needed. 
When the urine must be transported a long distance or 
kept more than a day or two a preservative may be re- 
quired. Formalin, one drop to four ounces of urine, will 
prevent decomposition. If more is added the formalin may 
reduce copper solutions used in sugar tests. 

Physical Properties. 

I. Amount. The normal quantity for an adult is from 
1000 to 1500 ex. in twenty-four hours. Children pass more 
in proportion to body weight than adults. A new born 
infant passes 150 to 200 c.c. A child of 5 years about 
700 c.c. 

In health more is passed during the day than during the 
night. This may be reversed in disease, notably in chronic 
interstitial nephritis. 

The amount varies physiologically according to the 

43 



44 URIN E 

amount of fluids and watery foods taken and the activity 
of other organs of elimination. 

Pathological factors influencing the amount are : 

1. The condition of the renal parenchyma. For ex- 
ample there is oliguria or temporary anuria in acute ne- 
phritis. 

2. The circulation in the kidney. The amount of urine 
is influenced by the rapidity of the blood flow, as well as 
by blood pressure. For example, there is oliguria in chronic 
passive congestion. Weak heart from any cause produces 
oliguria. Most diuretics increase the amount of urine by 
improving the circulation. 

3. Abnormal quantity or quality of substances excreted. 
The sugar in diabetes causes polyuria. At the beginning 
of convalescence from acute fevers, accumulated waste 
products stimulate the kidney to increased output of urine. 

4. Nervous causes. In hysteria there may be anuria, 
oliguria or polyuria. In various other nervous diseases 
variations in amount occur. The cause is thought to be 
vasomotor. 

5. The unknown cause of diabetes insipidus produces 
polyuria. 

II. Appearance. 

Normal urine is transparent, though a slight cloud of 
mucus may appear on standing. Cloudiness is generally 
due to pus, blood, bacteria, urates or phosphates. Albu- 
min itself even in large amounts causes no change in the 
appearance of the urine. The consistency of normal urine 
is watery, pathologically it may be frothy from the pres- 
ence of albumin, syrupy from the presence of sugar, or ropy 
from mucus or pus in alkaline urine. 



MANUAL OF LABORATORY DIAGNOSIS 45 

III. Color. 

Normal urine is amber or straw colored. It may vary a 
great deal within normal limits. 

Pathological Changes of Color : 

1. Blue or green. The administration of methylene 
blue usually accounts for a blue or greenish color. Rarely 
a bluish urine appears from putrefactive changes in the in- 
testines in cholera. 

2. Dark yellow or greenish brown color is usually due 
to bile 

3. Black. Melanin from a melanotic sarcoma may color 
the urine black. In salol, carbolic acid or iodoform pois- 
oning, the urine may turn black on standing. 

4. Red, brownish or smoky color may be due to blood. 

5. Red color in alkaline urine may be due to phenol- 
phthalein, rhubarb, senna or cascara. 

6. Milky color is found when there is an admixture of 
chyle. 

IV. Reaction. 

Normal urine is usually acid in reaction but may be 
temporarily neutral or alkaline. An amphoteric reaction, 
the urine turning red litmus paper blue, and blue paper red 
is not unusual, and has no pathological significance. Alkaline 
urine may be due to decomposition in the bladder as in 
cystitis with retention of the urine, or to admistration of 
alkaline drugs. A meat diet has the tendency to increase 
the acidity, and fruit and vegetable diet to diminish acidity. 

The reaction is determined by litmus paper, acid urine 
turning blue litmus red, and alkaline urine turning red 
litmus blue. 



46 URINE 

Total Acidity. — 

The total acidity of urine varies considerably normally. 
The average normal acidity is 400° to 600°. In estimating 
the total acidity the specimen of urine should be a mixture 
of the twenty-four hour output and should be as fresh as 
possible. 

Folin's Method of Estimation. — 

Measure 10 c.c. of urine into a 100 c.c. flask, add about 25 
c.c. of distilled water, 10 drops of 1% alcoholic solution of 
phenolphthalein and about 2 grams of potassium oxalate. 
Add slowly N/10 NaOH until a permanent faint pink 
color appears. The flask should be shaken after each 
addition of alkali and the alkali added drop by drop after 
the first one or two c.c. has been allowed to run in. The 
number of c.c. of N/10 NaOH required to neutralize 1 c.c. 
of urine multiplied by the number of c.c. of urine passed 
in 24 hours gives the degree of total acidity. 

V. Specific Gravity. 

The normal specific gravity of a 24 hour specimen of 
urine is between 1.012 and 1.020. Normally a great vari- 
ation occurs in separate passages of urine, the night urine 
having a high specific gravity. 

Physiologically the specific gravity varies inversely as 
the quantity. It is increased by nitrogenous diet. Patholog- 
ically the specific gravity is high in the following diseases : 

1. Diabetes mellitus. 

2. Acute nephritis. 

3. Acute fevers. 

The specific gravity is low in : 
1. Diabetes insipidus. 



MANUAL OF LABORATORY DIAGNOSIS 47 

1. Chronic interstitial nephritis. 'The specific gravity 
is markedly fixed and low, due to inability of the kidney 
to excrete a concentrated urine. 

The specific gravity is estimated 1))' means of the Squibbs 
urinometer. Dr. W. S. Harpole's Practical Urinometer en- 
ables one to take the specific gravity of a very small amount 
of urine. 

Total Solids. — 

By the total solids is meant the solids excreted in 24 
hours. The chief value of the estimation of the specific 
gravity is to give an idea of the amount of solids excreted. 
The normal total solids of an adult on ordinary diet is 
about 60 grams. About 4% of the weight of the urine is 
that of the solids and of this urea forms a little less than 
half. 

The total solids are estimated by the use of Haeser's 
coefficient. The last two figures of the specific gravity 
multiplied by 2.33 (Haeser's coefficient) equals the grams 
per litre. This multiplied by the number of litres passed 
in 24 hours gives the total solids. Example : 1400 c.c. of 
urine is the 24 hour amount. The specific gravity is 1.016. 
16x2.33x1.4=52.19. The total solids are 52.19 grams. 

Chemical Composition of Normal Urine. 

The 24 hour output consists of: 

1. Water, 1000 to 1500 c.c. 

2. Solids, 60 grams. 

a. urea, 30 grams. 

b. chlorides, 15 grams. 

c. sulphates, 2.5 grams. 

d. phosphates, 2.5 grams. 



48 URINE 

e. ammonia, 0.7 gram. 

f. uric acid, 0.7 gram. 

g. traces of numerous other substances. 

The amount of water and the various solids varies within 
wide limits in health, the above figures only serving to indi- 
cate the average output. 

Abnormal Substances Found in Solution in Urine. 

1. Proteids. Serum albumin, serum globulin, albumo- 
ses, nucleo-albumin, Bence-Jones proteid, mucin. 

2. Carbo-hydrates. Glucose, lactose, rarely other 
sugars. 

3. Acetone bodies. Acetone, diacetic acid and oxy-bu- 
tyric acid. 

4. Bile. 

5. Indican. 

Albuminuria. 

True albuminuria is the presence in the urine of albumin 
which has escaped through the cortex of the kidney. The 
conditions in which true albuminuria occurs are classified 
as follows : 

I. Albuminuria with definite renal lesions (usually 
large amounts of albumin). 

1. Nephritis in all its forms. 

2. Chronic passive congestion. 

3. Acute congestion of the kidney. 

II. Albuminuria without definite renal lesions (usually 
small amounts of albumin). 

1. Functional. After severe exercise, after prolonged 
cold baths, in the new born, cylic albuminuria. 



MANUAL OF LABORATORY DIAGXOSIS 49 

2. Febrile. 

3. Haematogenous. In severe anaemias and cachexias, 
and in chronic diseases as lues. 

4. Nervous. Usually transitory. 

False or accidental albuminuria is the presence in the 
urine of albumin from the admixture of pus or blood out- 
side the kidney substance, as in cystitis or injuries to the 
ureter, bladder or urethra. 

Serum albumin and serum globulin are usually both 
present in albuminuria, sometimes also nucleo-albumin and 
albumoses. Most ordinary tests respond to all these sub- 
stances. The appearance of proteoses or globulin except 
in association with serum albumin is rare. The Bence- 
Jones proteid appears in the urine in multiple myeloma. 
When present there is usually a large amount. This 
proteid coagulates on heating at about 60° C, clears up on 
further heating and reappears on cooling. 

For all albumin tests the urine must be clear. If cloudy, 
filter. If still cloudy, mix the urine with light magnesia 
(magnesium oxide) and filter. 

Qualitative Tests for Albumin. — 

Heller's Test. Take an inch of urine in a test tube, un- 
derlay this with half an inch of nitric acid. If the urine is 
alkaline, acidify it with a few drops of dilute acetic acid 
before making the test. 

Interpretation of the test : 

Precipitates. 

1. Albumin shows a white ring at junction of acid and 
urine. 

2. Nucleo albumin or mucin shows white ring above 
the contact with clear zone between. 



50 URINE 

3. Urates show a cloud above the contact line which 
disappears on heating or on using diluted urine. 

4. Oleo-resins show a milkiness at the contact. They 
are not coagulated by heat. 

Colors. 

1. Normal — varies from faint pink to brownish ring 
at contact. 

2. Biliary pigment — dark green or blue. 

3. Indican — violet or bluish when great excess is present 

4. Iodides — intense brownish red. 

5. Anilin compounds — red to purple. 

Robert's Modification of Heller's Test. Use in place of 
nitric acid a reagent consisting of saturated aqueous solu- 
tion of magnesium sulphate 100 c.c. and 20 c.c. of nitric acid. 

The interpretation is the same as that of Heller's test so 
far as the precipitates are concerned and the test is more 
delicate. 

Purdy's Test for Serum Albumin. — 

Take 1/2 test tube full of clear urine, add 1/6 its volume 
of saturated solution of sodium chloride and 5 drops of 
50% acetic acid. Boil the upper portion. A cloud in the 
boiled portion indicates serum albumin. 

Quantitative Tests for Albumin. 

Purdy's Centrifuge Method. — 

Place 10 c.c. of urine in a graduated centrifuge tube. 
Add 3 c.c. of a 10% solution of potassium ferrocyanide, and 
2 c.c. of 50% acetic acid. Mix by turning, let stand 5 
minutes, then centrifuge three minutes at 1500 revolutions 
per minute. Read the percentage of the precipitate. 
Each mark represents 1%. The percentage by weight is 
approximately 1/50 of the bulk percentage as estimated by 
this method. 



MANUAL OF LABORATORY DIAGNOSIS 51 

Tsuchiyas modification of the Esbach method. — 

Use Esbach's albuminometer. Fill the tube to the mark 
U with urine. Add the reagent to the mark R. Mix by 
turning and let stand 24 hours. The scale reads in grams 
of albumin per litre of urine. To find the percentage move 
the decimal point one place to the left. If the precipitate 
comes up to the mark 4 the percentage is 0.4. 

Tsuchiva's Reagent : 

Phosphotungstic acid, 1.5 grams. 
Concentrated HO, 5 c.c. 
95% alcohol q.s. 100 c.c. 

Glycosuria. 

Glycosuria is the presence of glucose in the urine. The 
assimilation limit for glucose is the minimum amount of 
sugar the ingestion of which is followed by sugar in the 
urine. The assimilation limit may be tested by giving 100 
grams of glucose dissolved in )A pint of water two hours 
after a breakfast of bread and coffee. Two hours later 
test the urine. If sugar is present the assimilation limit 
is pathologically lowered. 

The assimilation limit is lowered in the following condi- 
tions : diabetes mellitus, pregnancy, starvation, acute dis- 
eases, exophthalmic goitre and destructive lesions of the 
liver and pancreas. 

Spontaneous permanent glycosuria is nearly always due 
to diabetes mellitus. 

Lactose is the only other sugar often found in the urine. 
This may appear when the function of lactation is inter- 
rupted, sometimes during lactation. 



52 URINE 

Qualitative Test for Glucose. — 

Haines' Test. — 

Boil an inch of Haines' solution in a test tube, add 5 
drops of urine, bring to a boil ; if no reaction occurs add 
5 more drops of urine and bring to a boil again. If sugar 
is present a yellow or reddish yellow precipitate is thrown 
down. Avoid doubtful results by not using too much urine 
nor boiling too long. A slight greenish precipitate often 
appears on standing in urine containing no sugar. 

Haines' Solution : 

Cupric sulphate, 12 grams. 

Potassium hydroxide, 45 grams. 

Glycerine, 90 c.c. 

Water q.s. 1000 c.c. 

Quantitative Test for Glucose. 

Haines' Method. — 

Measure 10 c.c. of Haines' quantitative sugar test solu- 
tion into a 100 c.c. flask and add 50 c.c. of water. Dilute 
the urine by adding 4 parts of water to 1 part of urine and 
fill graduated burette with diluted urine. Bring the 
Haines' solution in the flask to a boil. Allow the diluted 
urine to run from the burette into the boiling solution very 
slowly and finally drop by drop until the blue color dis- 
appears. 10 c.c. of Haines' solution is decolorized by 
0.01 gram of glucose, therefore, whatever amount of urine 
was used to decolorize the Haines' solution in the flask 
contained 0.01 g. of glucose. 0.01 divided by the number 
of c.c. of undiluted urine used would give the grams of 
sugar in each c.c. of urine. 100 times this would give 
the amount in 100 c.c. or the per cent of sugar. 

Example : 1 c.c. of urine decolorized the fluid in the 
flask. 0.01-1-1X100=1=% of sugar. If the 24 hour amount 



MANUAL OF LABORATORY DIAGNOSIS 53 

of urine was 3000 c.c. the grams of sugar excreted in that 
time would be 1% of 3000 or 30 grams. 

Haines' Solution for Quantitative Sugar Determination : 

Copper sulphate 8.314 grams 

Potassium hydroxide 25 grams 

Ammonia 350 c.c. 

Glycerine 40 c.c. 

Distilled water q.s 1000 c.c. 

The Acetone Bodies. 

B-Oxybutyric acid, diacetic acid and acetone are spoken 
of as the acetone bodies. One or more of them will ap- 
pear in the urine when they are in excess in the blood. 
Poisoning by these bodies is called acidosis. This acidosis 
occurs when there is carbohydrate starvation. The 
source of the acetone bodies is the fats. The ammonia out- 
put in the urine is increased in proportion to the output of 
acetone bodies. The system is protected against the in- 
creased acidity due to the acetone bodies by neutraliza- 
tion with ammonia. 

This sort of acid poisoning may occur, 1st, when car- 
bohydrates are not ingested, as in starvation ; 2nd, when 
carbohydrates cannot be retained or digested, as in can- 
cer of the stomach and some intestinal disorders ; 3rd, 
when carbohydrates cannot be assimilated, as in diabetes 
mellitus. 

Test for acetone. (Gunning's) — 

To Y* test tube of urine add about 1 c.c. each of tincture 
of iodine and ammonia. A black precipitate of nitrogen 
iodide forms and gradually disappears. If acetone is 
present iodoform is formed, and is recognized by its odor, 
yellow color and the microscopic appearance of its crystals. 



54 U R I N E 

Test for diacetic acid. ( Gerhardt's ) — 

To y 2 test tube of urine add a few drops of tincture of 
ferric chloride. If a precipitate (phosphates) occurs filter 
and add more ferric chloride to the filtrate. A dark red 
color which fades somewhat on boiling or on standing sev- 
eral hours, indicates diacetic acid. Salicylates, phenol and 
antipyrine cause a dark red color which does not fade on 
boiling. 



Urea. 



Normally about 85% of the nitrogen excreted in the 
urine is in the form of urea. This proportion is altered 
in acidosis because the nitrogen is taken up in forming 
the ammonia which combines with the acid. 

Urea is increased when there is, 

1. Increased intake of nitrogen, as with meat diet. 

2. Increased tissue destruction, as in fevers or after 
excessive exercise. 

Urea is decreased when there is, 

1. Decreased intake of nitrogen in the food. 

2. Destructive disease of the liver, as acute yellow 
atrophy. 

3. Renal insufficiency as in acute nephritis and chronic 
interstitial nephritis. 

Test for Urea. — 

Use Doremus' ureometer. Remove albumin from urine 
if more than a trace is present, by acidifying with acetic 
acid, boiling and filtering. Fill the long arm of the ure- 
ometer with freshly made sodium hypobromite. Add slowly 
through the curved pipette exactly 1 c.c. of the urine. The 
gas evolved is nitrogen and bears a constant relation to 



MANUAL OF LABORATORY DIAGNOSIS 55 

the amount of urea present. The percentage of urea is 
read on the scale. 

Sodium hypobromite is made by adding 1 c.c. of bromine 
to 30 c.c. of a 20'/ solution of sodium hydroxide. The 
Doremus ureometer is filled with the 20^ sodium hydrox- 
ide and the bromine introduced with a curved 1 c.c. pipette. 

Ammonia. 

The normal excretion of ammonia in the urine is less 
than one gram in twenty-four hours. 

Notable increase in ammonia occurs in acidosis, the acid 
bodies being combined with ammonia to protect the tissues 
against the acids. In pernicious vomiting of pregnancy 
ammonia is much increased. The estimation of it may 
help to distinguish between pernicious vomiting and ner- 
vous vomiting. When the urea forming function of the 
liver is interfered with more of the nitrogenous waste 
appears as ammonia and correspondingly less as urea. 

Formalin Method of Estimation. 

To 10 c.c. of urine in a 100 c.c. flask add 25 c.c. of dis- 
tilled water and 5 drops of a 1% alcoholic solution of phe- 
nolphthalein. Neutralize by the addition of N/10 NaOH. 
In a second flask measure 5 c.c. of formalin, add 25 c.c. 
of water and neutralize with N/10 NaOH, using phenol- 
phthalein as an indicator. Add the neutralized formalin to 
the above neutralized urine. The resulting mixture will 
become colorless because the formalin breaks up the am- 
monium salts and liberates the acids. Titrate this acid in 
the mixture with N/10 NaOH. The number of c.c. N 10 
NaOH used in this last titration X0.0017 will give the 
number of grams of NH :J in 10 c.c. of urine. From this the 
amount in 24 hours can be calculated. 

Example: Suppose 4 c.c. N/10 NaOH was used to 



56 URINE 

neutralize the acidity after adding- the formalin. 4X0.0017= 
0.0068 grams of ammonia in 10 c.c. of urine. If 1000 c.c. 
is the amount of urine passed in 24 hours 0.0068X100=0.68 
grams ammonia in 24 hours. 

Uric Acid. 

The source of the uric acid is the nucleins. It is present 
normally as urates of sodium, potassium and ammonium. 
It crystallizes in the urine when in excess, or when the 
urine is very concentrated and acid. The variations in 
amount are considerable in health. 

Uric acid is increased : 

1. After ingestion of foods rich in nucleins, as sweet- 
breads or liver. 

2. In leukaemia, from the breaking down of many nu- 
cleated cells. 

3. During and after the paroxysm in gout. 

4. In fevers, corresponding to the increase in urea. 

Uric acid is decreased : 

1. In advanced kidney disease. 

2. Preceding the paroxysm in gout. 

Test for uric acid. — 

Use Ruhemann's uricometer. Introduce through a pipette 
carbon disulphid up to the mark S. Add the iodine mixture 
to the mark J. Mix well. The carbon disulphid becomes 
a deep red color. Add urine up to the lowest calibration, 
mix by turning. Add more urine, a few drops at a time, 
turning after each addition, until the carbon disuJphid i.3 
white. The reaction is then complete. Read the mark on 
the scale even with the surface of the urine. The scale 
reads in grams of uric acid per litre of urine. 



MANUAL OF LABORATORY DIAGNOSIS 57 

Ruhemann's reagent : 

Iodine, 0.5 g. 

Potassium iodide, 1.25 g. 

Absolute alcohol, 7.5 c.c. 

Glycerine, 5 c.c. 

Distilled water q.s., 100 c.c. 

Indican. 

Indol is a product of decomposition of proteid in the in- 
testine. It is oxidized in the blood to indoxyl, combines 
with sulphuric acid to form indoxyl sulphate of potassium 
(indican), in which form it is excreted in the urine. An 
excess appears in 

1. Intestinal putrefaction, 

a. Due to intestinal indigestion, diarrhoea, intes- 
tinal tuberculosis, constipation, etc. 

b. When HC1 is lacking in the stomach as in 
chronic gastritis or cancer. 

c. When there is a lack of peristalsis as in peri- 

tonitis or ileus. 

2. Putrefactive processes elsewhere in the body, as em- 
pyema, lung abscesses, advanced pulmonary tuberculosis. 

Obermayer's Test. — 

Pour y 2 inch of urine into a test tube, add an equal amount 
of Obermayer's reagent and about 1 c.c. of chloroform. Mix 
well. If an excess of indican is present the chloroform 
will be colored blue, the depth of the color depending upon 
the amount of indican present. 

Obermayer's Reagent : 

Hydrochloric acid, 500 c.c. 
Ferric chloride, 1 gram. 



58 • URINE 

The HC1 breaks up the potassium indoxyl sulphate and 
frees indoxyl. The ferric chloride oxidizes the indoxyl 
to indigo, which colors the chloroform blue. 

In urine containing bile or in any highly colored urine 
a doubtful color may appear in this test. This is avoided 
by removing disturbing substances with lead acetate. To 
half a test tube of urine add 1/5 its volume of a saturate 
solution of lead acetate. Let stand a few minutes, filter 
and test filtrate. 

Other oxidizing agents can be used with HC1 in detect- 
ing indican. 

The following test is more delicate than Obermayers 
and shows a faint color with normal urine : 

Pour 5 ex. of HC1 into a test tube, add 1 drop of HNO' 
and 15 drops of urine. Mix well. If indican is present an 
amethyst color appears, reaches its maximum in from 5 to 
10 minutes and then changes to yellow. 



Bile. 



Bile in the urine is always pathological. It appears 
whenever bile is present in the blood, that is, in jaundice 
from any cause. Bile may appear in the urine before the 
jaundice is apparent in the skin. Bile salts and bile pig- 
ments usually appear together and tests for bile pigments 
usually suffice for both. 

Tests.— 

Urine containing bile has a very yellow or greenish color, 
it foams when shaken and the foam is distinctly yellow. 
It stains filter paper yellow. The formed elements in the 
sediment are stained vellow. 



MANUAL OF LABORATORY DIAGNOSIS 50 

Gmeliris Test. — 

Layer the urine with nitric acid. If bile is present 
a dark green color will be seen at the contact. 

Smith-Rosen Test. — 

Layer 2 c.c. of Smith's reagent upon an inch of urine. 
Emerald green ring appearing at the contact indicates bile. 

Smith's reagent : 

Tincture of iodine, 10 c.c. 

95% alcohol, 90 c.c. 

The Diazo Reaction of Ehrlich. 

This reaction never occurs in health, and rarely in non- 
febrile diseases. 

It is nearly always present in typhoid fever and measles, 
occasionally in pneumonia, scarlet fever, diphtheria, ery- 
sipelas and tuberculosis. 

This reaction occurs in about 80% of the cases of ty- 
phoid, appearing early, often during the first week, and 
disappearing with the subsidence of the fever. Its reap- 
pearance during a relapse may serve to distinguish relapse 
from complication. The reaction is more constant and 
more marked in severe cases. 

A positive Diazo assists in distinguishing measles from 
rotheln. The appearance of a positive Diazo in tubercu- 
losis indicates a bad prognosis. 

Test : 

To 40 parts of solution I and one part of solution II 
(two fingers of solution 1+4 drops of solution 2) in a test 
tube add an equal amount of urine. Add quickly an 
excess of ammonia and shake. A deep red color and a pink 
foam constitute a positive reaction. A dark greenish pre- 
cipitate appears within 24 hours. 



60 URINE 

Solution I : 

Sulphanilic acid, 1 gram. 

Hydrochloric acid, 5 c.c. 

Distilled water, 100 ex. 
Solution II : 

Sodium nitrite, 0.5 grams. 

Distilled water, 100 c.c. 

The solution of sodium nitrite should be freshly made. 

Sediments in Urine. 

Sediment in urine is never strictly normal but may not 
be of serious significance. 

Methods of examination. A few facts may be obtained 
by macroscopic examination. Urates and phosphates may 
be recognized by appearance and chemical reactions. Uric 
acid in large amount is visible to the naked eye and char- 
acteristic in appearance. For microscopic examination the 
urine is centrifuged, the liquid poured off, the last drop 
in the centrifuge tube is shaken, then poured upon a slide, 
covered with a cover glass, and examined first with low 
power, afterwards with high, if it contains small bodies not 
easily identified with the low power. The light should 
be considerably cut down with the iris diaphragm as hyaline 
casts and other transparent bodies will be entirely invisible 
if there is too much light. 

Unorganized or Chemical Sediments. Those which have 
no cellular form and no connection with the cellular ele- 
ments of the body. 

1. In acid urine. 

A. Amorphous. 

Urates, a heavy cloud usually pinkish or brick 
dust color, soluble on warming. 

B. Crystalline. 

a. Uric acid. Amber, rarely colorless, large crystals 
of various shapes, rhombic plates, diamonds, 
"whetstones" and "butcher's block" or short 



MANUAL OF LABORATORY DIAGNOSIS 61 

cylinders. They tend to form in groups and 
masses or rosettes. They are soluble in caustic 
soda and insoluble in hydrochloric or acetic acid. 
They appear when uric acid is in excess and the 
urine is very acid and concentrated. They are 
only significant when found in a freshly voided 
specimen. They may form renal or vesical cal- 
culi, and may be found in the urine together 
with blood cells in this case. 

b. Oxalate of calcium. Small, colorless, refractile. 
Common form is the envelope. Sheaf, hour 
glass and oval forms may appear. They vary 
much in size. They dissolve in strong hydro- 
chloric acid. They appear after a diet rich in 
oxalates, as strawberries, rhubarb, tomatoes or 
spinach. They often accompany intestinal in- 
digestion and muscular pains. They may form 
calculi and masses of them with blood cells may 
be found in this case. 

c. Cystin. Colorless, six sided plates. They are 

rare. They are due to abnormal proteid metab- 
olism and individuals sometimes have them in 
the urine throughout life. They form calculi. 

d. Lencin and tyrosin. They appear together, the 
leucin in yellowish balls, the tyrosin as needle 
crystals in sheaves They are rare, being found 
in acute yellow atrophy of the liver and phos- 
phorous poisoning. 

2. In alkaline urine. 
A. Amorphous. 

a. Phosphates. Granular whitish cloud. Dissolves 

on the addition of acids. 

b. Carbonates. Indistinguishable from phosphates 
in appearance. Dissolves on addition of acids 



62 U R I N E 

with «erTervescence. They indicate decompo- 
sition of the urine. 

B. Crystalline. 

a. Ammonium-magnesium phosphate (triple phos- 

phate). Colorless, usually large, prism forms. 
The "coffin lid/ 5 "boot jack'' and occasionally 
feathery forms are seen. They are soluble in 
acetic acid. They usually signify nothing but 
decomposing urine, but when found in fresh 
specimens they point to alkaline fermentation in 
the bladder. 

b. Ammonium urate. The only urate which appears 

in alkaline urine. Brown prickly balls, "thorn 
apple" crystals, often grouped. They are soluble 
in acetic acid with formation of uric acid crys- 
tals. They appear only in decomposed urine 
containing free ammonia and have no pathologi- 
cal significance. 

c. Calcium carbonate. Small indefinite "dumb bells" 

associated with amorphous phosphates. 

Organized or Anatomical Sediments. — 

1. Epithelial cells. Some are always present. The por- 
tion of the urinary tract from which the cells come can- 
not be definitely located. They are of three general types. 

a. Squamous. Large, thin, flat, leaf like cells, often 

in sheets. Rather small round nucleus. From 
bladder, urethra or vagina. 

b. Irregular cells. Caudate, pyramidal, cylindrical. 
May come from deep layers of epithelium any- 
where in the tract. 

c. Round cells. Small round cells with compara- 

tively large nuclei, often slightly granular. These 
include the renal cells, but cells similar in 
appearance may come from other portions of the 
tract. Such cells are not common in normal urine. 



MANUAL OF L4B0RAT0RY DIAGNOSIS 63 

In nephritis they are often seen to be in a state of 
fatty degeneration. 

2. Red blood cells. Small round, homogeneous, non- 
nucleated cells. 'They may retain their normal disc shape 
and pale yellow color or they may be crenated, or swollen 
and colorless (shadow cells). Blood cells are always path- 
ological. Their source cannot be determined by appear- 
ance but may sometimes be suggested. 

Sources of blood : 

a. Acute nephritis and exacerbations of chronic 

nephritis. From an occasional cell to large 
amounts. Urine usually smoky colored. 

b. Malignant disease of the kidney or bladder. Hem- 
orrhage almost constant and may be profuse. 

c. Renal tuberculosis. Blood is not constant but is 

often found. 

d. Calculus, after the passage of a stone from the 
kidney or bladder. 

• e. Acute cystitis. Urine usually red. 

f. Polypoid tumor of bladder. Often causes profuse 
hemorrhage. 

g. Acute urethritis. 

h. Traumatism from catheter. 

i. Poisons, as turpentine, carbolic acid, cantharides 
and urotropine (occasionally after long administra- 
tion). 

j. Acute infectious diseases, as yellow fever, malaria 
and small pox. 

k. Hemorrhagic diseases, as scurvy, leukemia, purpura 
and haemophilia. 

3. Pus cells. About twice as large as red blood cells, 
round, granular, with irregular nuclei indistinct or invisible. 
They may be much or little degenerated. The addition of 
acetic acid to the slide will clear I'p the nuclei and make 



64 URINE 

their characteristics more distinct. An occasional leuco- 
cyte may be found in normal urine. 

Sources of pus : Urethritis, cystitis, pyelitis, from 
various bacterial causes, including gonococci and tubercle 
bacilli. In nephritis a few pus cells may be present. 

4. Casts. They are molds of kidney tubules. They 
are cylindrical, finger shaped bodies, of varying length. 
They appear when the kidney is affected by nephritis, circu- 
latory changes or toxic irritants. The same causes which 
produce albuminuria cause casts. They are usually found 
together, though either may be found without the other. 

The following varieties are recognized : 

a. Hyaline. These are homogeneous and so transpar- 
ent that the light must be considerably cut down 
in order to see them. They vary much in size, 
some being very small. They appear with less 
provocation than other varieties of casts, being 
found in temporary irritation of the kidneys, after 
anaesthetics, etc. They appear also in connection 
with other varieties in all forms of nephritis, and 
may be the only cast found in advanced interstitial 
nephritis. 

b. Granular. The substance of this cast appears finely 
or coarsely granular and more or less dark look- 
ing. Sometimes the base of the cast seems to be 
hyaline with granules partly filling the clear sub- 
stance. Granular casts indicate a definite lesion of 
the kidney and seldom appear except in nephritis. 
The coarsely granular cast has a more serious sig- 
nificance than the finely granular. 

c. Waxy. Homogeneous, but less transparent and 
more refractile than hyaline. They are usually 
large, sometimes very long. Their color is some- 
times slightly yellowish. They are of stiffer con- 
sistency than hyaline casts as shown by their ten- 



MANUAL OF LABORATORY DIAGNOSIS 65 

dency to break off square at the end, and the fact 
that they are not seen to bend. They are some- 
times twisted and often show transverse cracks. 
They appear in advanced nephritis and are of seri- 
ous significance. In acute nephritis a cast called 
by many writers "fibrinous" is occasionally found 
which is difficult to distinguish from waxy. It is 
apt to be yellowish or brownish in color. 

d. Epithelial. The cells of the tubules compose or 
cover these casts. Sometimes a hyaline or granu- 
lar base appears. Sometimes the cast seems en- 
tirely formed of epithelial cells. They are found 
in acute and chronic nephritis and point to a seri- 
ous lesion of the kidney. 

e. Blood cell casts. These casts are covered by or 
formed of red blood cells. They appear whenever 
there is exudation of blood into the kidney tubules, 
oftenest in acute nephritis. 

f. Pus casts. A few leucocytes may be attached to 
casts of any variety but true pus casts are formed 
entirely of pus cells. They are found in pyelo- 
nephritis, and are always of grave significance. 

g. Fatty casts. These consist of masses of fat 
globules. They are the product of fatty degenera- 
tion of epithelial cells composing epithelial casts. 
If the degeneration is only partial and the cells 
can still be made out they usually are called epi- 
thelial. They appear in both acute and chronic 
parenchymatous nephritis. 

Pus, blood and epithelial cells may all be found in the 
same cast. It will take its name from the predominating 
cells or be called a mixed cell cast. 

Cylindroids are bodies of the apparent composition of hya- 
line casts, only less solid. One end appears to be molded 



66 URINE 

in a kidney tubule and the other end trails off into a nar- 
row thread. They have but little significance, appearing 
often in normal urine as well as in company with hyaline 
casts. 

Narrow, long, transparent, microscopic threads of mucus 
are a frequent finding in urine. Their source is the bladder 
and their significance nothing. These should not be con- 
fused with mucous shreds (tripperfaden) which are 
opaque flecks or strings seen by the naked eye, often a 
centimeter long, and when viewed under the microscope 
are seen to be thickly studded with leucocytes. 

5. Bacteria. A few of the varieties which cause infection 
of the urinary tract can be identified without cultural meth- 
ods, as the tubercle bacillus and the gonococcus. Normal 
urine is sterile but is a good culture medium, therefore all 
specimens not obtained and kept in an aseptic manner rap- 
idly grow bacteria which have no significance but which 
cause cloudiness of the urine. 

6. Spermatozoa. Spermatozoa may appear in the urine 
of adult males after intercourse or nocturnal emissions, as 
well as in cases of spermatorrhea and after epileptic or 
other convulsive attacks. 

7. Yeasts and moulds are usually contaminations, but 
have been known to infect the bladder in cases of dia- 
betes, the saccharine urine furnishing a favorable medium 
for their growth. 



MANUAL OF LABORATORY DIAGNOSIS 



67 



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blood cells few or many. 
A few leucocytes. 


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especially granular, fatty and 

waxy. Occasional blood cells. 

A few leucocytes. 


Hyaline and granular casts. 

May be but few. Occasional 

red blood cells. 






Pus in small or large amounts. 
Blood may be present. 
Tubercle bacilli. 


Red blood cells, few or many. 

Pus cells if there is pyelitis. 

Crystals of calcium oxalate. 

Uric acid or cystin. 


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Very large amount of al- 
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Albumin present, often in 

small amounts. May be 

temporarily absent. 


Sugar present. 

Acetone bodies in severe 

cases. 


No albumin nor sugar. 
Normal solids in normal 
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Albumin present, a trace 
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Cloudy or smoky. 

Specific gravity high. 


Small or normal amount. 

Normal or dark in color, 

cloudy. 

Specific gravity high or normal. 


Large amount, light color, 

clear. 

Specific gravity low. 


Very large amount. 

Color very light, may be 

greenish, clear. 

Specific gravity very high. 


Very large amount. 

Color very light, clear. 

Specific gravity very low. 


Amount normal or large. 
Cloudy, acid reaction. 


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CHAPTER IV. 
STOMACH CONTENTS. 
Test Meal.— 

A specified test meal removed at a definite time is nec- 
essary so that the findings may be comparable. The 
Ewald breakfast is usually used in this country and con- 
sists of 35 grams of bread (2 slices) and 400 c.c. (2 cups) 
of water or clear weak tea. The test meal is given on an 
empty stomach and the stomach contents are then removed 
in 1 hour. 

Stomach contents after a test meal normally consist of 

1. acids; hydrochloric acid, free and combined, 

2. ferments; pepsin or pepsinogen, rennin or rennin zy- 
mogen ; 

3. food products; starch and products of starch diges- 
tion, i. e., erythrodextrin and achroodextrin; proteids and 
products of proteid digestion, i. e., acid albumin, albumose 
and peptone ; and acid salts. 

Pathological substances which may be present are lactic 
acid (more than a trace), mucus (excess), blood, bile, pus, 
bacteria (large numbers), yeasts and moulds. 

Examination of Stomach Contents 
After Ewald Test Breakfast. 

I. Macroscopical Examination. — 

Observe amount, color, odor, character of food particles 

69 



70 STOMACH CONTENTS 

whether coarse or finely divided, consistency whether 
watery or viscid, mucus whether mixed with food or float- 
ing*. If mucus is in excess and mixed with the food the 
stomach contents will string out when poured from one 
container into another. 

II. Microscopical Examination. — 

Place a drop of unfiltered stomach contents on a slide, 
add a drop of Lugol's solution, mix, cover with cover glass 
and examine with high dry lens. Starch granules are 
stained blue or violet, epithelial cells, pus cells, yeast cells 
and bacteria, yellow, and fat droplets are unstained. 

III. Chemical Examination. — 

Filter and use the filtrate for the chemical examinations. 
1. Titration. 

Measure 5 c.c. of filtrate into each of two flasks. Dilute 
each to 25 c.c. with distilled water. 

To the first flask add 3 drops of phenol phthalein (1 % 
alcoholic solution) and 1 drop of dimethylamido-azo-benzol 
(0.5 % alcoholic solution). If free HC1 is present, solution 
turns red; if free HC1 is absent solution turns yellow. To 
the second flask add 1 drop of alizarin (1 % aqueous solu- 
tion). If free acid is present solution turns bright yellow, 
if absent solution turns violet. 

Titrate mixture in flask No. 1 with N/10 NaOH from 
burette until red color changes to orange. Take burette 
reading. Number of c.c. N/10 NaOH used X20 indicates 
degree of acidity due to free HC1. Continue titration until 
first permanent pink color appears. Take burette read- 
ing. Total number of c.c N/10 NaOH used, including the 
amount used in first determining the free HC1X20 indi- 
cates the degree of total acidity. 



MANUAL OF LABORATORY DIAGNOSIS 71 

Titrate solution in flask No. 2 until yellow color changes 
to violet. Take burette reading-. Number of c.c. N/10 
NaOH used X20 subtracted from degree of total acidity 
indicates degree of combined HC1. 

The sum of the free and combined HC1 subtracted from 
the total acidity, gives the degree of acidity due to acid 
salts and organic acids. 

The acidity of stomach contents is usually expressed in 
degrees, degree of acidity meaning the number of c.c. of 
N/10 NaOH required to neutralize the acidity of 100 c.c. 
of stomach contents. 

Phenol phthalein indicates total acidity. Dimethyl- 
amido-azo-benzol (Topfer's Reagent) indicates free HC1. 
Alizarin indicates all acidity except that due to combined 
HC1. 

2. Test for Ferments.— 

If hydrochloric acid is absent test for ferments. Pepsin 
and rennin run parallel, therefore the test for rennin being 
simpler is sufficient for both. 

Pour 5 c.c. of milk into each of 3 t. t. and add 2 c.c. of 
1 % solution calcium chloride to each. Dilute 1 c.c. of 
filtrate from stomach contents with 9 c.c. of water, and add 
5 c.c. of this dilution to tube 1. To the remainder of the di- 
luted filtrate add 5 c.c. of water, and add 5 c.c. of this sec- 
ond dilution to tube 2. Incubate the three tubes J / 2 hour. 

If rennin is present in normal amount the milk in tubes 
1 and 2 will be coagulated. If the milk is not coagulated 
in either tube rennin is absent. If the milk is coagulated 
in tube 1 and not in tube 2. rennin is deficient. 

3. Test for Lactic Acid. — 

Lactic acid is never found in the presence of free HO, 
hence test for it only when free HO is absent. 

Kelling's Test. To a t. t. full of distilled water add ferric 
chloride solution to just color the water. Divide into 2 t: t. 



72 STOMACH CONTENTS 

Add filtrate from stomach contents drop by drop to one of 
the tubes, the second tube being used as a control. If lactic 
acid is present a canary yellow color will appear as drops 
are added. 

4. Test for Blood.— 

Weber's Test. Add 1/3 volume glacial acetic acid to tin- 
filtered stomach contents (about 10 c.c.) and shake. Add 
about 5 c.c. ether and gently invert tube a few times. Add 
1 c.c. of fresh tincture of guaiac (alcoholic solution of gum 
guaiac), and excess of hydrogen peroxide (about 2 c.c). If 
blood is present a blue ring will appear at the line of con- 
tact of ether and stomach contents spreading upward 
throughout the ether. The depth of color indicates roughly 
the amount of blood present. 

5. Test for Peptone. — 

Add a few drops of Haines' solution to about 5 c.c. of 
filtered stomach contents. A violet color will appear if 
peptone is present. Peptone is present if combined HC1 is 
present. 

6. Test for Starch.— 

Add Lugol's solution drop by drop to about 5 c.c. of fil- 
tered stomach contents. A blue color will appear if un- 
changed starch is present, a brownish color if erythrodex- 
trin is present and no change in color or an absorption of 
the color of the Lugol's solution if achroodextrin is pres- s 
ent. Normally after the addition of the first drops of Lu- 
gol's solution there is some absorption of color due to the 
presence of achroodextrin followed later as more Lugol's 
is added by the appearance of the brownish color due to 
erythrodextrin. 



MAX UAL OF LABORATORY DIAGXOSIS 73 

Normal Stomach Contents After Ewald Test Breakfast. 

Macroscopic: 

Amount, less than 150 c.c. 

Color, grayish white to yellow. 

Character of food particles, finely divided. 

Odor, sour. 

Consistency, semi fluid, separates into two layers. 

Mucus, little or none mixed with food. 

Chemical : 

Total acidity. 40 : -60 . 

Free HC1. 20'-40\ 

Combined HC1, 10 : -20'. 

Organic acids and acid salts, less than 10''. 

Pepsin and rennin present. 

Peptone present. 

Erythrodextrin and achroodextrin present. 

Lactic acid absent. 

Blood absent. 

Microscopic : 

Few bacteria. Xo Boaz-Oppler bacilli, sarcinae or 
yeasts. Few epithelial cells. Xo pus or blood 
cells. Mucus from the throat is often found in the 
stomach contents and shows numerous leucocytes 
and epithelial cells. Numerous starch granules 
stained blue or violet with Lugol's solution. 

Pathological Variations. 

Amount. — Is increased in hypersecretion and motor in- 
sufficiency'. 

Color. — Blood may give a red, brown or black color. 
Green color may be due to bile or green algae. 



74 STOMACH CONTENTS 

Character of Food Particles. — Coarse when HC1 is de- 
ficient or absent. Remnants of former meals found in 
stasis. 

Consistency. — Watery in hypersecretion, thick and viscid 
when excess of mucus is present. 

Mucus. — If intimately mixed with food is pathogno- 
monic of mucous gastritis. If in lumps or floating in liquid 
portion, it is swallowed mucus. 

Free HC1. — Increased in hyperacidity and most cases 
of gastric ulcer. Decreased in acute gastritis, chronic gas- 
tritis, and general systemic depression. Absent in most 
cases of carcinoma, advanced chronic gastritis and perni- 
cious anemia. 

Lactic Acid. — Present only in absence of free HC1. Its 
presence is the most valuable single diagnostic symptom of 
gastric cancer. 

Ferments. — If ferments are absent it is due to actual de- 
struction of the secreting glands. They are seldom absent 
except in atrophic gastritis and carcinoma. 

Starch Digestion. — Hyperacidity inhibits starch diges- 
tion, therefore in hyperacidity unchanged starch is present. 
If HC1 is diminished or absent, starch digestion proceeds 
to achroodextrin. 

Proteid Digestion. — Proteid digestion is poor when there 
is no free HC1 and little combined HC1. Little or no pro- 
teid digestion takes place if total acidity is very low. Pro- 
teid digestion is rapid in hyperacidity. 

Blood. — Usually present in carcinoma (coffee ground). 
Hemorrhages occur in many cases of ulcer. Occult blood 
(chemical, not visible) at times in ulcer, as a rule in car- 
cinoma. Gastric hemorrhages may occur (a) in primary 
disease of the stomach as ulcer or cancer, (b) secondary 



MANUAL OF LABORATORY DIAGNOSIS 75 

disease of other organs as in chronic passive congestion 
and cirrhosis of the liver. 

Bacteria. — Large numbers of bacilli, micrococci and yeast 
cells are present when there is stasis. Yeasts and sarcinae 
are present in gastric dilatation of benign origin. In gas- 
tric carcinoma large numbers of large, long bacilli, the 
Boaz-Oppler bacilli, are present. 



76 



STOMACH CONTENTS 



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CHAPTER V. 
FECES. 
Macroscopical Examination. — 

Observe color, whether "tarry," red or streaked with 
blood, whether colorless, glistening gray or clay-like from 
excess of fat or absence of bile. Normally there is a great 
variation in color dependent chiefly upon the diet. 

Observe the excess of mucus, whether in the form of 
shreds and flakes mixed with feces, or in ribbon like strips, 
or mixed with blood or pus. 

Examine washed feces for stones, connective tissue and 
parasites. To wash feces, place a small amount in a large 
beaker, add water, stir, let settle, pour off water and re- 
peat process several times. 

Microscopical Examination. — 

Make three separate mounts of feces, (1) one drop of 
feces mixed with water for examination of food remnants, 
blood, pus, ova and parasites as protozoa, (2) one drop of 
feces mixed with one drop of Lugol's solution for identifi- 
cation of starch granules, (3) one drop of feces mixed with 
one drop of Sudan III (70% alcoholic solution) for identifi- 
cation of fat. Examine under both low power and high dry 
lens. 

Food Remnants. 

1. Muscle fibres. Nearly always present. Appear as 

77 



78 FECES 

cylindrical yellow fragments, somewhat rounded, striated 
or homogeneous according to the degree of digestion. 

2. Vegetable detritis. Always present and generally in 
the form of plant cells with a distinct cell wall, commonly 
starch masses enclosed in a cellulose membrane, thorn like 
spines from fruits or berries, spiral cells (veins of leaves), 
pitted ducts. The background is made up of masses of 
bacteria and formless debris. Free starch granules are 
stained blue or violet with Lugol's solution. 

3. Neutral fat. Droplets or masses with irregularly 
rounded outlines stained red with Sudan III. 

4. Fatty acids. Small formless bile stained masses, or 
colorless delicate needle like crystals, often in sheaves. 

5. Soaps. Angular amorphous masses usually bile 
stained, or colorless short broad needle like crystals, often 
in sheaves. 

Mucus, Blood, Pus. 

Microscopically mucus appears as more or less transpar- 
ent masses with faintly marked outlines, usually mixed 
with food detritis, bacteria, leucocytes, epithelium and is 
often bile stained. Epithelium, leucocytes and blood 
cells are usually so altered and mixed with the feces they 
are hard to identify unless present in excess with mucus as 
in catarrhal, ulcerative, or dysenteric processes. 

Parasites and Ova. 

In examining for protozoa the feces should be fresh, the 
spread of feces on the slide thin and the slide warmed or 
kept on a warm stage in order to observe the movements 
of the parasites. The general recognition of the para- 
sites, their movements, etc., and of ova is done best under 
low power and further identification made under high dry 
lens. 



MANUAL OF LABORATORY DIAGNOSIS 79 

An amoeba should not be diagnosed unless its motility 
by pseudopodia can be demonstrated. At rest it resembles 
a large epithelial cell. Trichomonas intestinalis and lamb- 
lia intestinalis often associated with amoebae are very mo- 
tile, darting about by means of flagellae, and are about 3 
times the size of a red blood corpuscle. 

Most ova are oval in shape, yellow to brown in color, av- 
erage about 50x30 fx in size (size of many vegetable masses 
in feces), possess a definite shell and a central protoplasm 
which may be granular, segmenting or may contain an em- 
bryo. Vegetable cells are often mistaken for ova. The very 
definite form of the ova with their clean cut contour, the 
structure and thickness of their shells are usually sufficient 
to identify them. 

Chemical Examination. — 

Weber's Test for Blood. 

To about 5 c.c. of feces in a large test tube add y$ vol- 
ume of glacial acetic acid, cork and shake tube. Add 10 
c.c. of ether and invert the tube gently 3 or 4 times. To 
ether extract add 0.5 c.c. of fresh tincture of guaiac and 2 
c.c. of hydrogen peroxide. A blue color indicates blood. 
If fat is present in excess extract with ether several times 
before applying the test. 

If the feces has given a positive reaction or if occult gas- 
tric or intestinal blood is suspected it is best to put the 
patient on a meat free diet for a few days. If the reaction 
is then positive the blood is pathological. 

Pathological Findings and Significance. — 

Mucus. 

Any amount of visible mucus is abnormal. The 
amount seen pathologically varies enormously. It may be 
seen in the form of: 



80 FECES 

a. Shreds, lumps, small flakes, somewhat homogene- 
ous and transparent, rich in cells and detritis of 
digestion, varying in amount from small portions 
to nearly pure mucus, as in acute and chronic ca- 
tarrhal enteritis. 

b. Large amounts of mucus mixed with blood and 
pus, as in dysentery. 

c. Strips of tough leathery mucus from the large 
bowel as in conditions of secretory neurosis. 

Blood. 

The feces may be red or "tarry" from the presence of 
blood, may contain microscopic blood or occult blood, the 
color of the blood containing feces depending upon the 
amount and source of blood. 

1. Blood in streaks on formed stool points to hem- 
orrhoids or rectal fissure. 

2. "Tarry stool." Blood usually comes from stomach 
or duodenum. Blood in large amounts from small 
intestine with increased peristalsis may appear red 
as in hemorrhage from typhoid or duodenal, ulcer. 

3. Occult blood. Continuous in malignant diseases 
of the alimentary tract. Occasional and intermit- 
tent in peptic or duodenal ulcer. 

4. Blood mixed with pus and mucus in dysentery. 

Pus. 

Pus in feces is usually indicative of ulceration. Large 
amounts of pus may appear in case of ruptured extra-intes- 
tinal abscesses, extensive ulcerated carcinomata of colon 
or rectum, and dysentery. Small amount of pus is present 
in the feces in the majority of cases of simple ulcer. 



MANUAL OF LABORATORY DIAGNOSIS 81 

Fat. 

A clay-like stool usually contains fat droplets and large 
masses of fatty acid crystals. 

An excess of fat in the feces may occur when there is : 

a. Increased peristalsis. 

b. Interference with fat absorption in small intestine 
as in amyloid degeneration of the intestine, tuber- 
culosis of the intestine, chronic tubercular peri- 
tonitis, tabes mesenterica, cancer of the intestine. 

c. Biliary obstruction. 

d. Pancreatic disease. 

Stones. 

1. Gall stones. These may be found in the feces after 
the colic in cholelithiasis. They are friable, yellow or 
brown, smooth or facetted, and vary much in size. They 
are composed of bilirubin, calcium and cholesterin, and show 
concentric layers when fractured. 

2. Pancreatic stones (rare). In size no larger than a 
pea, usually single, colorless, irregular in shape, composed 
of calcium carbonate and calcium phosphate. 

3. Enteroliths or fecal concretions (rare). Undigested 
masses impregnated with calcium and magnesium phos- 
phate, very hard, may reach the size of large egg. 



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in feces. 

Worms in feces. 

Ova about the 

anus. 

Ova in feces. 


Ova in feces. 

Worm rarely in 

feces. 


Rhabditiform 

or filariform 

embryos in 

feces. Ova and 

adult forms 

rare. 


z 

O 

h 
o 
iu 
u. 

z 


re 

> 
O 


Ova. 


Larvae enter 

intestine 

through food 

or dirt or 

burrow 

through skin. 


Ova. 


Little known. 

Probably 
through em- 
bryo. 


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Embryo set free from ovum in 
small intestine, becomes sexually 
mature as it passes along the in- 
testine. Pregnant females in colon. 


Ova develop in dirt or feces out- 
side the intestine to larval form. 
Larval form enters intestine, con- 
tinues development in small in- 
testine and attaches itself to mu- 
cous membrane. 


Adult worm develops in same form 
from ovum. 


Life history complicated. From ova 
develop the rhabditiform embyro 
which generally passes into filari- 

orm embryo which develops intc 
pathenogenetic females (strongy- 
loides intestinals) and sexually ma- 

ure (Rhabditis stercoralis.) 


< 

h 

m 
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(0 

■h re s - 
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.. E 

— v re 
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Large and 

small 

intestine. 


Small 
intestine. 


Large 
intestine. 


Small 
intestine. 


LU 

N 

St 

O 

Ll 


Round worm 4-10 

inches long. 

Brownish color, 

characteristic odor. 


Round worm, '4 
inch long. 


Round worm, ]/ 2 

inch long. Whitish 

or blotched with 

brown. Hooklets 

about mouth. 


Round worm, 2 

inches long. Ant. 

end like thong of 

whip. Post, end 

like handle of 

whip. 


Embryos ]4 — Vz 
mm. Cylindrical, 
tapering to tail. 


LU 

< 

Z 


Ascaris 
-umbricoides 
(Stomach 

Worm) 


Oxyuris Ver- 

micularis 
(Pin worm or 
seat worm) 


Uncinaria 
duodenale. 
Anchylos- 
toma duode- 
nale (Hook 
worm) 


Trichocepha- 
lus trichiuris 
(Whip worm) 


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CHAPTER VI. 

HUMAN MILK. 

Composition of Normal Human Milk. 

Reaction — slightly alkaline or neutral. 
Sp. Gr.— 1028-1032. 
Fat— 3 to 5%. 
Lactose — 6 to 7%. 
Protein— 1 to 2.25%. 

Microscopically, fat droplets are present and during the 
first few days of lactation, colostrum corpuscles (large 
granular cells). 

Variations in the amount of fat, proteid and sugar are 
the chief changes which have a practical bearing on infant 
feeding. 

Fat. 

Use Holt's cream gauge. Fill cream gauge to zero mark 
with fresh milk. Let stand 24 hours. Read % of cream. 
The ratio of cream to fat is as 5 to 3. Thus 5% cream indi- 
cates 3% fat. 

The fat content may be determined immediately with 
the small Babcock tube which fits the cup of the ordinary 
centrifuge. 5 c.c. of milk is pipetted into the tube, 5 c.c. 
of concentrated H 2 SO is added a little at a time, mixing 
with each addition, and finally enough of a mixture of equal 
parts of concentrated HC1 and amyl alcohol is added to fill 

85 



$6 HI 'MAX MILK 

the tube. Centrifuge the tube 5 minutes and read the per- 
centage of fat directly on the tube. 

Protein. 

Boggs' Modification of the Esbach Method. 

Dilute the milk ten times and rill Esbach albuminometer 
to mark U with diluted milk. Add the reagent to the mark 
R. Mix by turning. Let stand 24 hours. With this dilu- 
tion the marks on the tube give the percentage of the pro- 
tein. 

Boggs' Reagent. 

Phosphotungstic Acid 25 grams. 

Concentrated Hydrochloric Acid 25 c.c. 

Distilled water q. s. ad 250 c.c. 

Lactose. 

Mix equal parts of milk and Boggs' Reagent, dilute with 
equal volume of water, filter and estimate lactose in the fil- 
trate by the titration method as in the quantitative esti- 
timation of sugar in urine. 0.014 grams of lactose re- 
duces 10 c.c. of Haines' quantitative solution. 

To calculate : 0.014 divided by the number of c.c. of 
undiluted milk used multiplied by 100 gives the % of lac- 
tose. 



CHAPTER VII. 

CEREBRO-SPINAL FLUID. 

Normal cerebrospinal fluid is limpid and colorless and 
contains very few cells. It contains enough sugar to re- 
duce copper solutions and a very slight trace of proteid 
material. 

Bacteriological Examination. 

Make smears from centrifuged sediment of spinal fluid. 
Stain with Gram, also with . carbol fuchsin if tubercle ba- 
cillus is suspected. Make cultures of centrifuged sediment 
on blood agar and blood serum, using culture tubes and 
Petri dishes in which blood agar has been poured. In 
smears and cultures examine for meningococcus intracel- 
lularis, tubercle bacillus, pneumococcus. streptococcus, ty- 
phoid bacillus, colon bacillus and influenza bacillus. Other 
bacteria have been reported but rarely. In examining for 
tubercle bacilli in spinal fluid it is best to allow the fluid 
to stand 24 to 48 hours to allow a pellicle to form. This 
pellicle may contain the bacilli when they cannot be found 
in the fluid proper. 

Cytology. 

Differential Cell Count. 

The fluid is centrifuged, the sediment smeared, dried and 
fixed on a slide and stained with Wright's blood stain. A 
differential count of the cells is then made as a differential 

87 



88 CEREBROSPINAL FLUID 

leucocyte count is made. In the acute infections the poly- 
morphonuclear cells predominate and in the chronic infec- 
tions as tuberculosis and syphilis the lymphocytes predom- 
inate. 

Total Cell Count. 

In fluids containing few cells (clear fluids) the number 
of cells per cu.mm. is estimated as follows : The leucocyte 
pipette is filled to the 1 mark with 10% acetic acid, the 
fresh spinal fluid, well shaken, is then drawn up to the 
mark 11. The mixture is shaken and a drop mounted in 
the counting chamber as in counting blood. The Tiirck 
counting chamber is used and the cells in the whole ruled 
space, 9 sq. mm., are counted. This space contains 9/10 
cu. mm., of fluid. The mixture is 9/10 spinal fluid and 1/10 
diluting fluid. Therefore the number of cells counted 
X10/9X10/9= the number of cells per cu. mm. For ex- 
ample the 9 sq. mm., contains 40 cells. 40X10/9X10/9= 
49+. If cells are so numerous as to cause clouding, the 
spinal fluid must be diluted as for a leucocyte count of the 
blood. The number of cells per cu. mm. of normal spinal 
fluid is less than 10. 



Chemical. 



Test for Globulin.— 



The Ross-Jones modification of the Nonne test for ex- 
cess of globulin is as follows : 1 c.c. of spinal fluid is care- 
fully layered upon 2 c.c. of saturated solution of ammonium 
sulphate in a small test tube. A grayish white ring at the 
contact, appearing within a few minutes, is a positive re- 
action and indicates a pathological amount of globulin in 
the spinal fluid. 

The test is positive in all infections of the nervous system 
and negative in normal spinal fluid. 



MANUAL OF LABORATORY DIAGNOSIS 89 

The Lange Colloidal Gold Reaction. 

Technique of the Test. 

Ten test tubes (6 in. x Y\ in.) are placed in a row in a 
test tube rack. To the first add 1.8 c.c. of a freshly made 
0.4% sodium chloride solution. To each of the other 
tubes add 1 c.c. of the same solution. Draw up into a 1 c.c. 
pipette 0.2 c.c. of the spinal fluid to be tested and add it to 
the first tube. This makes a dilution of 1-10 in tube 1. 
From the first tube remove with the same pipette 1 c.c. of 
the mixture and place it in tube 2. This will make a dilu- 
tion of 1 in 20 in tube 2. Remove 1 c.c. from tube 2 and 
place it in tube 3, making a dilution in tube 3 of 1 in 40. 
Continue in this way to the end of the series, discarding the 
1 c.c. removed from tube 10. The dilution in each tube is 
double that in the tube before it. the dilution in tube 10 
being 5120. Add to each tube in the series 5 c.c. of the 
colloidal gold solution. At the end of twenty-four hours 
a final reading is made, although strong reactions will 
show in a much shorter time. 

The reaction consists in color changes due to more or 
less precipitation of the colloidal gold. The solution itself 
is red and clear. Slight precipitation gives a bluish tint 
to the fluid. Increasing amounts of precipitation change 
it to violet blue, grayish and when precipitation is com- 
plete, colorless. The amount of change is expressed by 
numbers, 5 representing complete precipitation and the 
least observable change 1. A negative reaction would be 
written 0000000000. One in which precipitation is com- 
plete in the first four tubes and less in the fifth, sixth and 
seventh would be written 5555431000. Preparation of the 
colloidal gold solution used as indicator requires great care 
and is often unsuccessful in inexperienced hands. Specific 
directions for its preparation with discussion of its difficul- 
ties may be found in Bull. Johns Hopkins Hospital xxvi, 



90 CEREBROSPINAL FLUID 

1915, p. 391. The colloidal gold solution can be purchased 
from E. H. Sargent. 

Diagnostic Value. 

The diagnostic value of the test is considerable as it dis- 
tinguishes several types of cerebro-spinal disease. 

In general paresis complete precipitation occurs in the 
lower dilutions, the typical reading being 5555431000. Com- 
plete precipitation may occur in even higher dilutions. 

In cerebro-spinal syphilis and tabes the greatest color 
change occurs in the third and fourth tubes, a typical read- 
ing being 1133200000. 

In non-syphilitic meningitis the greatest precipitation oc- 
curs in the higher dilutions, that is, beyond the fourth tube. 

In normal spinal fluid the color of the whole series re- 
mains unchanged. 



MANUAL O/-' LABORATORY DIAGNOSIS 



< 

DC 
III 

h 
O 
< 
CO 


Meningococcus 
Pneumococcus 
Streptococcus 
Influenza B, etc. 


Tubercle 
bacillus. 


Spirochaeta 
Pallida 


Absent. 


DIFFEREN. 
TIAL CELL 
COUNT 


Polynuclears 
predominate 


Mononuclears 
predominate 


(0 « 

«1 

4) .E 

1 E 

O 0) 

o a 

2 




&3 

. _J 
O LU 

zo 


.e 

CI 

S- 
V 

> 


■o 

(0 

re 

0) 

o 

c 


■a 
<u 

10 

re 

D 

L. 

o 

c 


c E 
££ 

s| 

-Jo 


DC 
< 
O 
D 
(0 


c 

(0 

.a 
< 


Present. 
May be less 
than normal. 


re 

E 
s_ 
o 
Z 


Present. 
Reduces 
Fehlings. 


z 

J 

D 

m 

O 

o 


c • 

— T3 

n 

£ re 
o a* 


D 

eg 

re 

o 

c 


Usually 
Increased. 

Slight 

trace. 

Negative 

Nonne. 


DC 

< LU 
UJ O 
Q.Z 
£L< 
< 


May be clear, 

often cloudy, 

coagulates on 

standing. 


Clear. 
Delicate pellicle 
on standing. 


L. 

ro 

0) 

O 

i 


Clear and 
colorless. 


LU 

< 
LU 
(0 

Q 


(0 

If 

< «= 

2 


5S 

n 

11 


Syphilitic 
Diseases of Ner- 
vous System 


re 

E 

c 
o 
Z 



CHAPTER VIII. 
SPUTUM. 
Macroscopic. — 

The consistency or character of the sputum depends upon 
the presence and relative amount of mucus, serum, pus or 
blood present. Mucoid, serous, muco-purulent, purulent 
and sanguinous are terms descriptive of the character of 
the sputum as determined by its leading constituents. The 
more mucus there is the more tenacious the sputum. The 
color due to blood may be the color of fresh blood or that 
of the haemoglobin derivatives. 

Formed bodies which may be recognized macroscopically : 

a. Curshmann's spirals, twisted threads of mucus, 
characteristic of bronchial asthma. 

b. Dittrich's plugs, foul smelling, cheesy, cylindrical 
masses, found in chronic bronchitis. 

c. Elastic fibres and necrotic tissue found in advanced 
tuberculosis or other destructive diseases of the 
lung. 

d. Molds of the bronchi, found in fibrinous bronchitis. 

e. "Sulphur granules" found in actinomycosis. 

Microscopic. — . 

Unstained. A loopful of the sputum is put on a slide, cov- 
ered with a large cover glass and examined with high dry lens. 

93 



94 SPUTUM 

The ray fungus of actinomycosis, blastomycetes, hooklets of 
echinococcus, entamoeba histolytica and pigmented epithe- 
lial cells and crystals are best seen in the unstained speci- 
men. 

Stained. The most important information to be gained 
from the stained specimens is the demonstration of the tu- 
bercle bacillus, and other pathogenic bacteria. The standard 
method for the demonstration of the tubercle bacillus in spu- 
tum is the Ziehl-Neelsen Method, which is described under 
stains in the chapter on bacteriology. A Gram stain should 
be made for the examination of bacteria other than the tu- 
bercle bacillus. The chief pathogenic bacteria to be looked for 
are the pneumococcus, the influenza bacillus, Friedlander's 
bacillus, micrococcus catarrhalis and streptococcus. When any 
of these bacteria are the causative agent of an infection they 
will be present in large numbers in the sputum and will be 
the predominating bacteria. 

Sputum in Certain Diseases. — 

Pulmonary Tuberculosis. 

Tubercular sputum is usually purulent, but sputum of any- 
character may be found in this disease. The presence of the 
tubercle bacillus is the diagnostic feature. The more ad- 
vanced the destructive process in the lung, the greater the 
number of tubercle bacilli present. 

Acute Lobar Pneumonia. 

Early, the sputum is scanty, tenacious, translucent and rusty 
colored. Toward the crisis it becomes more abundant, muco- 
purulent and later during resolution becomes mucoid. If 
blood continues to be present, as it often does, the sputum 
is colored dark by it. The pneumococcus is present in large 
numbers in those cases caused by the pneumococcus. 

Acute Bronchitis. 

Sputum is at first scanty, mucoid, soon becomes abundant 
mucG-purulent, then purulent, sometimes blood streaked. 



MANUAL OF LABORATORY DIAGNOSIS 95 

Chronic Bronchitis. 

Sputum usually abundant, muco-purulent, may vary much in 
character and amount. 

Asthma. 

Often there is no sputum during the paroxysm; if pres- 
ent it is scanty, clear, consisting of thick glairy mucous balls. 

Curshmann's Spirals, Charot-Leyden crystals, and eosino- 
phils are characteristic. 

Pulmonary Oedema. 

Large amounts of frothy, serous, blood stained sputum. 

Abscesses, Gangrene, Bronchiectasis, Tubercular Cavities. 

The intermittent appearance of large amounts of purulent 
sputum, often fetid, containing necrotic tissue, and crystalline 
products of decomposition is characteristic. 



CHAPTER IX. 

BACTERIOLOGY. 

Methods of Bacteriological Examination of Pathological 

Material. 

The bacteriological examination of pus or other material 
consists of (1) the examination of stained smears to determ- 
ine the morphology of the bacteria, the staining reactions, the 
spore and capsule formation, the number of bacteria and the 
distribution of the bacteria, whether intra or extracellular; 
(2) the cultural study to isolate the different varieties of bac- 
teria in pure culture and to identify them by cultural charac- 
teristics as to the appearance and luxuriance of growth on the 
different media, the production of chemical changes in the 
media such as the production of acid in milk, the fermen- 
tation of sugars w r ith acid or gas formation, the production 
of indol, the liquefaction of gelatin, of coagulated blood 
serum or of casein; (3) the serum reactions as the agglu- 
tination and complement fixation tests and (4) animal in- 
oculation. 

Smears and cultures should be made directly from the pa- 
tient as far as possible. When material is to be carried to 
the laboratory to be examined bacteriologically, whether in 
liquid form, tissue or on cotton swabs, it should be placed in 
dry sterile bottles. In aspirating pus or fluid, for example 
from tooth sockets, it is convenient to use capillary pipettes. 
These are made by drawing out tubing of the diameter 
used for the ordinary medicine dropper to capillary fineness 
of about 1 mm. They may be sterilized in the sterilizer or 

97 



98 BACTERIOLOGY 

in the flame. The capillary end is sealed in the flame 
after the fluid is aspirated, and when ready for examination, 
is broken off and pus blown out on slides for smears and 
on the surface of media in Petri dishes and tubes for 
cultures. 

. When only a small amount of pus is available platinum 
loops or cotton swabs (wooden applicators wound at the 
ends with a bit of cotton, sterilized in hot air sterilizer and 
stored in cotton plugged tubes) are more practical. Smears 
and cultures are made directly from the loop or swab. 

In examining extirpated tonsils make smears and cultures 
with a platinum loop from the depth of the crypts ; lay open 
the tonsils with a sharp sterile knife and make smears and 
cultures from the inside. The tonsil can be ground in a sterile 
mortar with a small amount of sterile salt solution after the 
surface baceria have been killed by plunging the tonsil into 
boiling water. Cultures are made from this salt solution 
emulsion. Extirpated glands may be ground up in salt solu- 
tion and examined in the same way. Urine, preferably a 
catheterized specimen, is centrifuged in sterile centrifuge tubes, 
the supernatant fluid poured off and the few drops of sediment 
used to make smears and cultures. Sputum collected in 
sterile bottles should be examined preferably soon after ex- 
pectoration. Smears and cultures should be made directly 
from the purulent portions of the sputum and also after wash- 
ing the sputum in several changes of sterile water to remove 
the surface mouth bacteria. Blood for bacteriological exam- 
ination is best obtained from one of the large veins at the bend 
of the elbow. The skin is thoroughly cleansed with alcohol, 
a constrictor applied to the upper arm, a sterile needle (large 
size, about No. 19) attached to a sterile syringe inserted into 
a vein and about 10 c.c. of blood withdrawn into the syringe. 
The needle is then removed and the blood distributed into 
flasks of glucose bouillon in the proportion of one part of 
blood to about 50 parts of bouillon. If growth takes place 
in the bouillon as shown by the microscopical examination of 



MANUAL ()[< LABORATORY DIAGNOSIS 99 

stained smears from the coagulum, transplants should be 
made on other media to identify the bacteria. 

Smears. 

For the making of smears one should have at hand slides, 
and a platinum loop or cotton swabs. A small loopful of 
the material to be examined or the cotton swab moistened 
with it is spread over a glass slide in a thin film, a loopful 
of water being added when necessary to aid in making the 
film thin. The smears are fixed in the flame and separate 
slides stained with methylene blue, Gram and carbol fuch- 
sin. If one slide only is used, Gram's stain is preferred. 
When several different films are to be stained in the same 
way parallel films can be made on one slide by using a 
small amount and stroking crosswise instead of lengthwise 
of the slide. 

Culture Media and Making of Cultures. 

The smears give some information as to the number and 
varieties of bacteria so that one can be guided as to the 
special media needed in making cultures and the amount 
of material to be used in inoculation. Culture media 
needed for general examinations : 

1. Agar agar — Petri dish and slant. 

2. Loeffler's blood serum — slant, aerobic and anaerobic. 

3. Blood agar — Petri dish and slant, aerobic and anae- 

robic. 

4. Ascitic glucose agar — slant and deep tube. 

5. Ascitic glucose bouillon. 

6. Glucose bouillon — fermentation tube. 

It is not always necessary to use all of these different 
kinds of media, but where possible it is best to do so as it 
gives a better chance to grow any bacteria present and a 
better separation of the colonies. If all media are not 
available use preferably blood agar and blood serum aero- 
bically and anaerobically. 



100 BACTERIOLOGY 

Loeffler's blood serum. — 

Mix one part of dextrose bouillon with three parts of 
beef serum, tube and sterilize the tubes on a slant in a 
blood serum inspissator or Arnold steam sterilizer on three 
successive days. 

Blood agar. — 

Melt about 10 c.c. of neutral or slightly alkaline glucose 
agar, cool to 50° C, add ^ to 1 c.c. of defibrinated blood 
(human or goat), mix and pour in sterile Petri dish to 
harden. Slant blood agar is made in the same way, slanted 
and allowed to harden. 

Ascitic glucose agar. — 

Melt glucose agar, cool to 45° or 50° C, add sterile ascitic 
fluid in the proportion of one part ascitic fluid to 3 parts 
of agar. Pour in Petri dish, slant in tube, or use for deep 
tube inoculation. 

Edno medium. (Kendall's modification.) 

1% lactose agar slightly alkaline to litmus, sterilized and 
stored. When needed add 1% decolorized fuchsin (1 c.c. 
sat. ale. sol. fuchsin+10 c.c. of 10% watery sol. sodium sul- 
phite), pour in plates, allow to harden .and inoculate by 
streaking. 

Bile Medium. (For typhoid blood cultures.) 

Add 10% of glycerine and 2% of peptone to ox bile. A 
10% solution of dried fresh ox gall can be used in place 
of the ox bile. The medium is sterilized in theautoclav and 
stored in flasks, 25 c.c. to a flask. 

In making a blood culture add 1 part of blood to 3 parts 
of bile medium. 

Inoculation of Media in Petri Dish. 

When the medium is sufficiently firm usually in 15 or 20 
minutes after pouring make many strokes across the plate 



MANUAL OF LABORATORY DIAGNOSIS 101 

with the platinum loop or swab dipped in the material to 
be examined. There will be a thick inoculaton under the 
first needle strokes and a thinning out of the bacteria under 
the last strokes, so that well separated colonies will appear. 
Incubate the plates with cover side down to prevent the 
water of condensation from mixing the colonies. 

Anaerobic Cultures. 

Anaerobic cultures are conveniently made on Loeffler's 
blood serum and slant blood agar, but may be made on any 
media. After inoculation the cotton plug is pushed down 
to the level of the top of the medium slant, the tube filled 
(as capsules are filled) above the cotton with pyrogallic 
acid to the depth of about 1 inch, 5 to 10 drops of 5% sodium 
hydroxide are added and a cork previously soaked in par- 
affin put in tightly. Lay the tubes in the incubator on a 
slight slant with the cork end downward. If anaerobic 
cultures are to be made in liquid media the same method 
is used, but an extra cotton plug is necessary to prevent 
the sodium hydroxide from working its way down to the 
medium, and the tube must be kept upright in the incu- 
bator. To inoculate deep tubes of ascitic glucose agar, melt 
the agar, add the ascitic fluid as previously described, then 
inoculate the medium while still fluid, mix well and let so- 
lidify. When solid make anaerobic in the same' manner as 
slant blood agar tubes. 

Stains and Methods of Staining. 

Methylene Blue Method.— 

1. Fix smear in flame. 

2. Cover with methylene blue j/> minute. 

3. Wash in water and dry between filter paper. 

Loeffler's Methylene Blue. (Wright's formula.). 

Methylene blue 0.5 gram. 

Sodium carbonate 0.5 gram. 

Water 100 c.c. 



102 BACTERIOLOGY 

Gram's Method. — 

1. Fix smear in flame. 

2. Stain ]/ 2 minute in anilin gentian violet. 

3. Wash in water. 

4. Cover with Gram's iodine y 2 minute. 

5. Drain and drop on 95% alcohol until alcohol runs 
off clear. 

6. Wash in water. 

7. Counterstain y 2 minute with pyronin (0.5% 
aqueous solution). 

8. Wash in water and dry between filter paper. 

Anilin- gentian-violet. 

Anilin water 75 c.c. 

Sat. ale. sol. gentian violet 25 c.c. 

Anilin water is prepared by adding 2 c.c. of anilin oil to 
98 c.c. distilled water, shaking the mixture vigorously for 
1 minute and filtering through filter paper until filtrate 
runs clear. 

Grains Iodine. 

Iodine 1 gram. 

Potassium iodide 2 grams. 

Water 300 c.c. 

The object of the iodine is not to stain and not to decol- 
orize, but to act as a mordant which sets the stain in some 
bacteria so that the alcohol will not decolorize them. 
Those bacteria in which the stain is set are Gram positive, 
i. e., they keep the gentian violet stain. Those bacteria 
which are decolorized by the alcohol are Gram negative 
and take the counterstain. In pus smears Gram positive 
bacteria appear dark purple. Gram negative bacteria and 
leucocytes stain pink. 



MANUAL OF LABORATORY DIAGNOSIS 103 

Ziehl-Neelsen Method. (Stain for tubercle bacillus.) 

1. In making smear from sputum, select purulent por- 
tion, pick up with wooden toothpick, smear on 

slide, fix in flame. Cover with carbol fuchsin. 
Keep steaming hot three minutes. 

2. Wash in water. 

3. Decolorize in 10% sulphuric acid in 95% alcohol 
until well spread portions are decolorized. 

4. Wash in water. 

5. Counterstain Yi minute in Loeffler's Methylene 
Blue. 

6. Wash in water and dry between filter paper. 

7. Examine with oil immersion lens. The tubercle 
bacilli show as slender red rods. Everything else 
in the sputum stains blue. 

Carbol fuchsin. 

Phenol crystals, melted 25 c.c. 

Absolute alcohol 50 c.c. 

Fuchsin (basic) 2 grams. 

Allow to remain over night in an incubator to insure 
complete solution, cool and filter. This stock solution is 
permanent and does not require further filtering. For use, 
add 1 part of this stock solution to 4 parts of distilled water. 

Diagnostic Characters of Pathogenic Bacteria. 

Staphylococci. 

Morphological and Cultural Characteristics. 

The varieties of staphylococci are differentiated on the 
basis of pathogenicity, pigment formation, liquefaction of 
gelatin and other cultural properties. They are named 
from their distinguishing characteristics, as Staphylococcus 



104 BACTERIOLOGY 

pyogenes aureus, Staphylococcus pyogenes albus, Staphy- 
lococcus citreus, Staphylococcus epidermidis albus, etc. 

Typically the pathogenic staphylococci are Gram posi- 
tive, appear in smears as spheres in grape-like clusters and 
as diplococci. Culturally they grow luxuriantly on all 
media, form moist elevated colonies, white, yellow or 
orange, depending upon the variety, liquefy gelatin freely, 
acidify and coagulate milk, and ferment sugars. 

Atypically staphylococci may vary in size from small 
to very large cocci, and may appear as flattened diplococci 
with a narrow space between the cocci. They may be 
Gram negative. They may grow in tiny colonies, or in the 
form of flat colonies with concentric markings, or they may 
form a film like adherent growth. They may liquefy gel- 
atin very slowly or not at all. 

Strains which liquefy gelatin freely are in general more 
virulent. Staphylococcus pyogenes aureus is more pyo- 
genic than the albus or citreus. 

Occurrence. 

The staphylococcus is the most common pus producer. 
It is found most frequently in skin infections as acne pus- 
tules, skin abscesses, furuncles, carbuncles. It may cause 
primary infection of sinuses and may cause septicemia. It 
is the most common cause of osteomyelitis. 

Streptococci and Pneumococci. 

Varieties. 

No absolute method has been found of differentiating the 
different varieties of streptococci from one another or from 
the pneumococcus, but a fairly satisfactory differentiation 
can be made on the basis of the capsule formation, the be- 
havior on blood agar, the ability to ferment the various 
sugars, the serum reactions (agglutination and complement 
fixation), and the pathogenicity towards animals. 



MANUAL OF LABORATORY DIAGNOSIS 



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106 BACTERIOLOGY 

There are many strains of streptococci found in the hu- 
man body and its secretions which cannot be readily classi- 
fied among the streptococci or pneumococci mentioned in 
the table, partly because of their morphology, partly 
because of their appearance on blood agar, but chiefly 
because of their lack of pathogenicity. Some are Gram 
negative. Some produce dry, brown adherent colonies on 
blood agar with very slight or no haemolysis or green col- 
or ization of the agar and are non-pathogenic towards ani- 
mals, or at least produce no visible lesion in heart or joints 
even in enormous doses. 

Pathogenicity. 

The virulence shown by the streptococci and pneumo- 
cocci varies considerably. The lesions produced by animal 
inoculations depend upon the virulence of the strain, the 
method of inoculation and the number of bacteria inocu- 
lated. The most virulent streptococci will cause fatal 
septicaemia, less virulent strains may cause localized ab- 
scesses, erysipelatoid inflammations or endocardial or joint 
involvement. Streptococcus hemolyticus tends to locate in 
the joints, while streptococcus viridans tends to locate in 
the endocardium. The pneumococcus and streptococcus 
viridans often show little virulence toward animals. 

Occurrence. 

Found in erysipelas, lymphangitis, cellulitis and puer- 
peral septicaemia, in suppurative inflammatory conditions 
in joints (as arthritis and acute rheumatism) and serous 
membranes (as peridicarditis and pleurisy), in otitis media 
and the throat affections of tonsillitis, diphtheria, scarlatina 
and measles, in enteritis in infants, and in pneumonia 
(pneumococcus in lobar pneumonia, streptococcus in the 
lobular pneumonia usually). 



MANUAL OF LABORATORY DIAGNOSIS 107 

Gram Negative Diplococcus Group. 
Gonococcus. (Diplococcus of Neisser.) 

Morphological and Cultural Characteristics. 

The gonococcus is a coffee bean shaped large diplococcus 
with flat sides adjacent. It is Gram negative. In pus 
smears the diplococci appear grouped in the cytoplasm of 
the pus cells. In freshly infected cases they as a rule are 
the only bacteria found. In chronic gonorrhea many other 
bacteria may be present and the microscopic diagnosis is 
not so simple. The gonococcus grows best on blood agar 
or ascitic agar in the form of delicate, fine, grayish white 
colonies. After continued cultivaton it may grow on 
ordinary media. Isolation of the gonococcus is only pos- 
sible from acute cases and even then it is very difficult. 

Occurrence. 

The gonococcus is found in the pus of acute gonorrhea 
and gonorrheal ophthalmia. Arthritis and endocarditis 
occur as metastatic complications. 

Micrococcus Catarrhalis. — 

Morphological and Cultural Characteristics. 

The micrococcus catarrhalis has the same morphology 
in general as the gonococcus, but is often slightly larger 
and tends to show a sharper outline. In pus smears it gen- 
erally stains more deeply than the gonococcus and is less 
often seen within the leucocytes. It grows well on ordi- 
nary media forming grayish white or yellowish white often 
adherent colonies of mortar like consistency. 

Occurrence. 

Found commonly in secretions of normal and diseased 
mucous membranes. 



108 BACTERIOLOGY 

Meningococcus. 

(Micrococcus intracellularis meningitidis.) 
Morphological and Cultural Characteristics. 

The meningococcus is a biscuit shaped micrococcus oc- 
curring usually in pairs, but may be seen also in fours or 
masses. Involution forms are common. It is Gram nega- 
tive and shows some irregularity in staining. In pus 
smears it is chiefly intracellular. It grows best on neutral 
ascitic glucose agar, forming flat, grayish white disk like 
colonies tending to become confluent. It may grow on 
agar after prolonged cultivation. It tends to die out in a 
few days after isolation. The different strains vary in the 
ease with which they can be grown on artificial media, in 
their fermentative action on sugars, and in their virulence. 

Occurrence. 

The meningococcus is the most frequent cause of puru- 
lent meningitis either sporadic or epidemic. In meningo- 
coccus meningitis the spinal fluid is cloudy and contains 
a large number of polynuclear leucocytes. The meningo- 
coccus is found chiefly within the cells. 

Bacillus of Tuberculosis. 

Morphological Characteristics. 

The tubercle bacilli are slender, straight or slightly 
curved rods often occurring in small clumps, the bacilli lying 
at an acute angle with one another. They vary in length 
and often show small nodules or swellings. They are 
acid fast, that is, they retain the fuchsin stain and are not 
decolorized by acids or alcohol as the non-acid fast bac- 
teria are. They commonly stain uniformly but may show 
a beaded appearance due to the deep staining of the nodules 
and the pale staining of the areas between. 



MANUAL OF LABORATORY DIAGNOSIS 109 

Diagnosis. 

Examination of. sputum for tubercle bacilli is commonly 
made. The usual and most satisfactory method is directly 
by stained smears. Care must be taken to obtain the 
sputum from the lungs. Purulent portions are smeared 
on slides, fixed in the flame and stained with carbol fuchsin. 
(See stains.) Thicker smears may be made in examining 
for tubercle bacilli than for the usual bacteriological exam- 
ination, thus permitting one to examine easily more ma- 
terial. One has no difficulty in distinguishing the red 
stained bacillus even in a thick smear. A careful exami- 
nation of more than one slide prepared from suspicious 
purulent portions will show the bacilli if they are present. 
The tubercle bacilli show as bright red rods against a blue 
background and show a characteristic arrangement and 
morphology. 

Tubercle bacilli in the urine are much more difficult to 
find than in sputum. Large amounts of urine must be 
centrifuged and the sediment smeared on slides, dried, 
fixed in the flame and stained as sputum smears. The pos- 
sible presence of other acid fast bacilli as the smegma bacil- 
lus rarely makes the diagnosis of tubercle bacilli in the 
urine uncertain. The examination of a catheterized speci- 
men will usually exclude the smegma bacillus. The only 
certain method is to inject a guinea pig with the sediment 
from a large volume of urine. 

Diphtheria Group. 

(Bacillus diphtheriae and the diphtheroid bacilli.) 

Morphological and Cultural Characteristics. 

The typical diphtheria bacillus produces a powerful 
toxin, grows rapidly and grows best on serum media, and 
shows a characteristic morphology in stained smears. The. 



110 BACTERIOLOGY 

diphtheria bacilli are slender rods which vary in length, 
are often slightly curved, often clubbed or swollen at the 
end or middle. They may lie at an acute angle with one 
another or they may lie in palisade arrangement. They 
are Gram positive. They do not stain uniformly with 
Loefller's methylene blue but show granules or barred 
staining. The pseudo-diphtheria bacilli tend to be short 
plump rods more uniform in size and shape, and generally 
do not show polar granules. The true and pseudo-diph- 
theria bacilli, however, cannot always be differentiated 
morphologically. 

Culturally the diphtheroid bacillus like the diphtheria 
bacillus grows best on serum media, but may grow well on 
all media. A valuable means of differentiating the Hof- 
mann bacillus or pseudo-diphtheria bacillus from the diph- 
theria bacillus is by growing the bacilli in glucose bouillon. 
The Hofmann bacillus does not produce acid by the fer- 
mentation of the glucose while the diphtheria bacillus does. 
It is not possible, however, to classify the numerous strains 
of diphtheria and diptheroid bacilli on the basis of their fer- 
mentative action on the various sugars. Diphtheroid bacilli 
of acne and those found in enlarged glands can be isolated 
anaerobically, but after cultivation may grow aerobically. 
The acne bacillus is easily and constantly isolated from 
acne pustules in anaerobic cultures on blood serum. 

Pathogenicity. 

The virulence of diphtheria bacilli varies considerably, 
but the majority are of nearly equal virulence. Diphtheria 
like bacilli are commonly found which are pathogenic to 
guinea pigs especially in enormous doses, but which pro- 
duce no diphtheria toxin. Animal inoculation is used as 
a test of virulence and as a test of toxin production. True 
diphtheria bacilli cause death in 72 hours of a guinea 
pig injected subcutaneously with a broth culture in amount 



MANUAL OF LABORATORY DIAGNOSIS 111 

less than 1/5% of the body weight. If a guinea pig in- 
jected with antitoxin lives after the injection of about 
2 c.c. of the broth culture of a bacillus and the control 
pig which received only the broth culture dies, one is deal- 
ing with a virulent diphtheria bacillus. 

Occurrence. 

Virulent diptheria bacilli are found in diphtheritic mem- 
branes, also occasionally on normal mucous membranes. 
Diphtheroid bacilli are commonly present on normal and 
inflamed mucous membranes as of the pharynx, nose, ear, 
eye (B Xerosis), urethra, vagina, etc., and on the skin as 
the acne bacillus in acne pustules. They are found in en- 
larged glands in Hodgkins disease, lymphatic leukemia, 
leprosy, etc. 

Diagnosis of Diphtheria. 

The diagnosis of diphtheria often rests upon the bacteri- 
ological findings and the early diagnosis is very important. 
The bacteriological diagnosis can often be made from a 
smear directly from the throat membrane. This should 
always be made as it may permit the administration of 
antitoxin many hours earlier. If the throat smear is doubt- 
ful or negative, wait for the examination of the culture. 
The culture is best made on blood serum and should be 
examined after 8 to 10 hours incubation as the diphtheria 
bacillus grows rapidly. Smears from the throat or smears 
from the culture should be stained with methylene blue. 
If the diphtheria bacilli are present they will show their 
characteristic clubbed form or their barred or orranule 



Hemoglobinophilic Bacillus Group. 

Occurrence. 

In this group belong the influenza like bacilli found in 



112 BACTERIOLOGY 

some epidemics of influenza, the pertussus bacillus found 
in whooping cough, the Koch-Weeks bacillus found in 
acute contagious conjunctivitis, the bacillus of Ducrey 
found in soft chancre, the influenza like bacilli found in 
diseases of the respiratory tract, in some cases of acute 
meningitis, measles and scarlet fever. The bacilli found 
in these various diseases belong to the group of hemoglo- 
binophilic bacilli and are identical morphologically and 
culturally, differing only in the matter of virulence and 
varying somewhat in that. 

Morphological and Cultural Characteristics. 

All the bacilli of this group require hemoglobin in the 
culture media for growth. They grow in tiny transparent 
colonies and die out quickly. They are very small, non- 
motile, Gram negative, bipolar staining bacilli. 

Bacterial Diagnosis. 

Differentiation between non-pathogenic influenza like 
bacilli found in sputum, etc., and this group of hemoglo- 
binophilic bacilli cannot be made in smears. Streak spu- 
tum on blood agar. Examine small shining colonies in 
stained smear to see if there are any influenza like bacilli. 
Transfer suspicious colonies to plain agar and blood agar. 
No growth will take place on plain agar if it is a true 
hemoglobinophilic bacillus. For further identification 
make agglutination test. 

Mucosus Capsulatus Group. 

This group includes B. Mucosus capsulatus (Bacillus ol 
Friedlander), B. lactis aerogenes, B. ozenae (Abel bacillus) 
and probably the Perez bacillus. 



MANUAL OP LABORATORY DIAGNOSIS 113 

Morphological and Cultural Characteristics. 

The bacilli of this group are non-motile, Gram negative, 
generally short broad bacilli but varying in their propor- 
tions and usually with prominent capsule. They grow well 
on all media, and form a viscid mucoid growth, the viscid- 
ity depending upon the degree of capsule formation. They 
generally ferment sugars with acid and gas production. 
There is a wide variation within the group from the bacilli 
of the colon type with no capsule to those with prominent 
capsule formation, from the bacilli growing like B. coli to 
the bacilli growing in mucus like masses and from the 
bacilli producing slight or no carbohydrate fermentation 
to those which ferment all carbohydrates with a large 
amount of gas production. 

Occurrence. 

The bacilli of this group are found constantly in the 
crusts and secretion of atrophic rhinitis and ozena, may be 
found in diseases of the middle ear and accessory sinuses 
of the nose, in lobular pneumonia, occasionally in cystitis, 
pyelitis, pericarditis, pleuritis and meningitis (secondary). B. 
lactis aerogenes is a normal inhabitant of the intestine es- 
pecially of children. 

Colon-Typhoid Group. 

This group includes the colon and paracolon bacilli, the 
typhoid and paratyphoid bacilli and the group of dysentery 
bacilli. 

Diagnostic Methods. 

These consist of (1) the isolation of the bacilli from the 
blood, feces, urine or other sites of infection, (2) the morph- 
ological and cultural study of the bacilli, and (3) for final 
identification the agglutination test, using immune sera 
against the various types. 



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MANUAL OF LABORATORY DIAGNOSIS 115 

Methods of Isolation of the Bacilli from Blood, Urine and 

Feces. 

From blood. Inoculate blood from the vein into broth in 
the proportion of 1 part of blood to 50 of broth, or into bile 
media in the proportion of 1 part of blood to 3 of bile. 
Identify by cultural characteristics and agglutination test 
with known immune sera. 

Front urine and feces. To isolate typhoid bacilli, streak 
plates of Endo medium with centrifuged urine sediment or 
a suspension of feces made by rubbing up feces with sterile 
water. By streaking several plates the inoculation can be 
sufficiently thin to have isolated colonies. Fish translu- 
cent dewdrop like colonies and make agglutination test with 
known typhoid serum. 

A probable diagnosis of colon bacilli in urine can easily 
be made by centrifuging catheterized urine in sterile centri- 
fuge tubes, and adding a few drops of sediment to a fer- 
mentation tube of dextrose bouillon. Colon bacilli will 
ferment the dextrose w r ith gas production 

Bacillus Proteus. (Putrefying bacillus.) 

Morphological and Cultural Characteristics. 

The proteus bacillus is an actively motile Gram nega- 
tive bacillus varying greatly in size, and ranging from 
short to long filaments. It grows on all media and best at 
room temperature, producing a putrefactive odor on blood 
serum and gelatin with liquefaction of the gelatin. The 
growth on gelatin plates is characteristic, consisting of an 
irregular radiating mass of colonies with liquefying center. 

Occurrence. 

The proteus bacillus is not uncommonly found in pye- 



116 BACTERIOLOGY 

lonephritis and cystitis. Meat poisoning may be due to 
bacilli of this group. Sometimes associated with other bac- 
teria as pus cocci or alone, the proteus bacillus has been 
found in purulent peritonitis and phlegmonous inflamma- 
tions. It may be found in the nasal secretion. 

Bacillus Pyocyaneus. (Bacillus of blue and green pus. ) 

Morphological and Cultural Characteristics. 

The pyocyaneus bacillus is very small, slender, actively 
motile and Gram negative. It grows well on all media, 
liquefies gelatin, and colors all media bright green in the 
presence of oxygen. 

Occurrence. 

The pyocyaneus bacillus may be found in inflammations 
of the serous membranes as of the pericardial sac and 
joints, of the mucous membranes as of the ear and sinuses 
of the nose. It has been found in broncho pneumonia and 
has been known to cause general infection. 

Bacillus Tetani. 

Morphological and Cultural Characteristics. 

Tetanus bacilli are motile, long slender rods usually 
single but often in long threads, form large round terminal 
spores and are Gram positive. They grow on ordinary 
media only under anaerobic conditions, producing an arbo- 
rescent growth on solid media. They liquefy gelatin and 
ferment sugars with gas production. They produce a 
powerful toxin. 

Occurrence. 

The tetanus bacillus is found in tetanus through wound 
infection. The bacilli multiply but little in the animal 
body and are usually associated with other bacteria, hence 
it is difficult to isolate them in pure culture. They produce 



MANUAL OF LABORATORY DIAGNOSIS 117 

a powerful toxin at the point of infection, and this spreads 
to the motor nerves by various channels, causing death. 

Diagnostic Methods. 

These consist of (1) the examination of smears from the 
pus of the wound, (2) the study of the cultures made from 
the pus or infecting" material and (3) animal inoculation. 

1. Make smears from the pus of the wound, stain with 
Gram, and examine for spored bacilli ; the tetanus bacillus 
is seldom found in the pus. 

2. Inoculate pus or bits of tissue or foreign bodies found 
in the wound into glucose bouillon and make anaerobic. 
Incubate the cultures 24 to 48 hours, then heat them j/ 2 
hour at 80° C. to kill all vegetative forms of bacteria. In- 
oculate the heated culture into glucose bouillon or milk, 
make anaerobic, incubate 24 hours and examine for the 
tetanus bacillus. 

3. To make certain test, inoculate mice or guinea pig 
subcutaneously with salt solution emulsion of material 
from the wound. Tetanus will result in 1 to 4 days if the 
tetanus bacilli or spores are present. 

Bacillus Anthracis. 

Morphological and Cultural Characteristics. 

Anthrax bacilli are large, non-motile rods with square 
cut ends. They grow in long chains, often in twisted 
bundles and show a prominent capsule. They are Gram 
positive. They form spores only in the presence of oxy- 
gen, hence not in the animal body. They grow on all media 
and liquefy gelatin. Their colonies are very characteristic 
and consist of a tangled mass of filaments. 

Occurrence. 

The anthrax bacillus is the causative agent in malignant 
pustule and intestinal and pulmonary anthrax. They may 



118 BACTERIOLOGY 

be recognized in- smears from the fluid of the malignant 
pustule, in the feces from intestinal anthrax and in the 
sputum from pulmonary anthrax. The diagnosis may be 
established by cultural stud}' and animal inoculation, the 
anthrax bacillus causing death of guinea pig by septicaemia. 

Bacillus. Aerogenes Capsulatus. (B. Welchi.) 

Morphological and Cultural Characteristics. 

The B. Welchi is a large non-motile bacillus growing 
singly and in chains. It forms a capsule when growing 
in the body and spores occasionally in cultures. It is Gram 
positive. Culturally it is a strict anaerobe and grows 
well on all media. It ferments the sugars with energetic 
gas production. It produces a characteristic growth 
(stormy fermentation) in milk, that is, it forms a much 
riddled clot because of the abundant gas formation. The 
milk is acidified by the formation of butyric acid which 
gives its characteristic odor to the culture. 

Occurrence. 

B. Welchi is common in the intestine and soil. It is the 
cause of emphysematous gangrene. 

Pathogenic Trichomycetes. 

Microorganisms of this group of higher bacteria grow 
in the form of long threads varying in length and thick- 
ness, some showing club like terminations, some branching. 
Among them there are Gram positive and Gram negative 
varieties, acid fast and non-acid fast varieties, aerobic and 
anaerobic varieties, some growing readily in artificial media, 
some grown with great difficulty. The growth on solid 
media is generally granular, adherent, dry like a mould, 
and in liquid media white, thistledown like tufts of inter- 
lacing filaments, or they may form a pellicle. They are 
widely distributed and not infrequently met with. They 
are of a low grade of pathogenicity, the usual reaction to 



MANUAL ()} ; LABORATORY DIAGNOSIS 119 

infection in man and animals being chronic granulation 
tumors with or without suppurative foci. 

Varieties. — 

Leptothrix. This grows in long, straight, thread like form 
and shows no branching. It is usually Gram negative. It is 
found frequently in the human mouth about the teeth and 
tonsils. It is of doubtful pathogenicity. 

Nocardia (Streptothrix). This grows in branching threacj 
like form and in smears appears as a tangled mass 
of threads. There are Gram positive and Gram negative 
strains. The growth is usually like that of a mould, but 
the different strains vary much in the ease with which 
they grow on artificial media. The anaerobic varieties 
generally show a scanty growth as compared with the 
aerobic. 

It may be found in skin abscesses, alveolar abscesses, 
brain abscesses, cerebrospinal meningitis, pneumonic areas, 
pseudotuberculosis of the lungs, etc. 

Actinomyces. This grows as branching threads. It is 
typically anaerobic. In the tissues the actinomyces grows 
in colonies in the form of yellow granules of about pin- 
head size. For diagnosis examine the pus or granulation 
tissue for these yellow granules. Crush the granules be- 
tween slides and examine unstained with high dry lens, 
also stain with Gram and examine with the oil immersion 
lens. The granules will be seen to be made up of a tangled 
mass of filaments tending to radiate from a center and 
showing characteristic club like terminations. The center 
of the colony may have a mass of coccus like bodies or 
conidia. The filaments and spores are Gram positive and 
the clubs usually Gram negative. 

Actinomyces causes the disease actinomycosis, which is 
rare in man. It may occur primarily in the mouth, head 
or neck, on the skin, in the lungs and in the intestine. 



120 BACTERIOLOGY 

Bacteriology of Conjunctival Secretions. 

The diagnosis of the bacteria in conjunctivitis can often 
be made by the examination of a Gram stained smear from 
the conjunctival secretion. In making a smear the dis- 
charge should be taken from the conjunctival surface 
avoiding" contaminations from the lid margins, etc. 
If the discharge is slight it is best to obtain what has 
collected at the inner canthus of the eye. Sometimes 
gentle pressure upward along the lachrymal duct will 
bring forth some discharge into the inner canthus 
of the eye. For collection of the discharge it is con- 
venient to use wooden applicators wound at the tip with 
a bit of cotton and sterilized. A platinum loop may also 
be used. Smears are made on clean slides by rubbing the 
pus collected on the cotton tip of the applicator or plat- 
inum loop into a thin layer on the slide. The smear is 
then stained with Gram's stain and examined with oil im- 
mersion lens. 

Koch-Weeks Bacillus. This may be found in acute con- 
tagious conjunctivitis. The bacilli are Gram negative 
slender rods of varying length. Morphologically they 
.cannot be distinguished from the influenza bacillus. 

Bacillus Influenzae. This may be found in catarrhal con- 
junctivitis. The bacilli are Gram negative very short rods, 
often resembling elongated diplococci. 

Morax-Axenfeld Bacillus. This may be found in subacute 
conjunctivitis. The bacilli are large Gram negative diplo- 
bacilli, some appearing in short chains. 

Bacillus Xerosis. This may be found on the normal con- 
junctiva. The bacilli are Gram positive diphtheroid bacilli. 

Pneinnococcus. This may be found in acute catarrhal con- 
junctivitis. The pneumococci are Gram positive, elongated 
often lancet shaped diplococci. The capsules are not seen 
in eye smears. 



MANUAL OF LABORATORY DIAGNOSIS 121 

Gonococcus. This may be found in purulent conjunctivitis. 
The gonococcus is a Gram negative biscuit shaped diplo- 
coccus, chiefly intracellular. 

Micrococcus Catarrhalis. This may be found occasionally 
on the normal conjunctiva and in simple catarrh. It is a 
Gram negative biscuit shaped diplococcus, generally extra- 
cellular and often in clusters. Generally the secretion is 
scanty and poor in cellular elements in proportion to the 
number of cocci. 

Meningococcus. This may be found in conjunctivitis ac- 
companying meningococcal meningitis. The meningo- 
coccus is a Gram negative biscuit shaped diplococcus, 
chiefly intracellular. 

Streptococcus. This may be found in severe membranous 
conjunctivitis. The streptococci are Gram positive spheri- 
cal diplococci and in chains. 

Bacteriology of Vincent's Angina. 

The diagnosis is easily and quickly made by examination 
of a smear from the exudate or membrane in the throat. 
The smear may be stained with methylene blue or Gram's 
stain. Spirilla and fusiform bacilli in large numbers, and 
a moderate number of leucocytes will be found in the 
smears. The spirilla are Gram negative, small and show 
shallow spirals. The bacilli are Gram negative, irregularly 
stained with methylene blue, are long, slender and spindle 
shaped. 

Cultures from the exudate on blood serum under anaero- 
bic conditions may yield a growth of the fusiform bacillus 
mixed with other bacteria. Such cultures have a foul odor. 

Preparation of Bacterial Vaccines. 

The preparation of an autovaccine consists in, (1) the 
obtaining of a bacterial culture, (2) the making of an emul- 
sion in salt solution of this culture, (3) the sterilizing of 
the emulsion, (4) the counting of the bacteria in the 



122 BACTERIOLOGY 

emulsion, and the diluting and bottling of the vaccine 
ready for use. 

Cultures. — 

The most difficult and most important step in the mak- 
ing of vaccines is the obtaining of satisfactory cultures. 
It is always advisable to first examine the smears made 
directly from the specimen, and to use this information 
about the varieties and numbers of bacteria present as a 
basis in selecting proper media and determining the amount 
of the material to be used in inoculation. To obtain cultures 
from abscesses, pustules or other pus containing a single 
organism, platinum loopfuls of the pus are smeared over 
the surface of 4 or 5 tubes of Loeffler's blood serum or plain 
agar, and incubated 24 hours. If there is a mixture of bac- 
teria in the specimen of pus, sputum, urine, feces, tissue, 
etc., from which the vaccine is to be prepared it will be 
necessary to separate the varieties of bacteria and get 
them in pure culture. To do this the material should be 
streaked over the surface of the media in Petri dishes of 
blood agar, over slant surface of a series of tubes of blood 
serum, and inoculated into liquid media as ascitic glucose 
bouillon, some of the inoculations being rendered anaero- 
bic. Pure cultures from the different bacteria can then be 
obtained from the single colonies and subcultures used in 
the preparation of the vaccine. Solid media, especially 
blood serum and blood agar, (5 to 10 tubes) is usually most 
practical for making the cultures for the vaccine, but in the 
case of streptococci and pneumococci more growth can be 
obtained in inoculation of about 50 c.c. of glucose bouillon or 
ascitic glucose bouillon. 

Preparation of the Emulsion. — 

If the cultures are on solid media add a few c.c. of sterile 
salt solution to each tube and gently scrape the growth 
from the surface with a platinum loop mixing with the 
solution. Pour the bacterial emulsion from each tube 



MANUAL OF LABORATORY DIAGNOSIS 123 

into a sterile centrifuge tube, centrifuge 2 or 3 minutes 
and pour off turbid supernatant fluid into sterile 25 c.c. 
bottle. Add a few c.c. of sterile salt solution to sediment 
in centrifuge tube, mix thoroughly by shaking, repeat cen- 
trifuging and again pour off supernatant cloudy fluid into 
same bottle with previous emulsion. This process can be 
repeated if necessary in order to get into an emulsion nearly 
all of the sediment, but usually twice is sufficient. This 
method gives a uniformly cloudy fluid, removes any clumps 
of bacteria and avoids the labor of prolonged shaking to 
break up the clumps. 

If the culture is in bouillon, this must be poured into 
sterile centrifuge tubes and centrifuged. The supernatant 
bouillon is then poured off and discarded, and sterile salt 
solution added in its place. The bacterial sediment is 
mixed well with the salt solution, the tube centrifuged and 
the supernatant fluid poured into a sterile bottle, the pro- 
cess being repeated if necessary until most of the bacterial 
sediment is in the form of a uniformly turbid emulsion. 

Sterilizing the Vaccine. — 

Sterilize the bacterial emulsion by keeping it at a tem- 
perature of 60° C. for one hour. This is done by placing 
the bottle containing the emulsion in a water bath at 60° 
C. allowing the water to come well up on the neck of the 
bottle. Any bacteria on the inside of the neck of the 
bottle are first killed by thoroughly flaming the neck of 
the bottle with Bunsen flame. 

Counting the Bacteria in the Emulsion. — 

Pour a few drops of the sterilized bacterial emulsion into 
a watch crystal. Have ready a clean dry watch crystal, 
a watch crystal with normal salt solution, three clean 
slides which have been passed slowly through the Bunsen 
flame 4 or 5 times to burn off grease, one capillary glass 
pipette with rubber teat fitted over the large end, and a 
few strips of cigarette paper cut the width of a slide. 



124 BACTERIOLOGY 

Mark the glass pipette with a glass pencil one inch from 
the tip. Stab finger with a needle and press out drop of 
blood. By suction with rubber teat draw up blood to pencil 
mark, draw in small measure of air then same measure of 
bacterial emulsion as blood, followed by 8 measures of salt 
solution each separated by air. Press all out in dry watch 
crystal, mix by drawing back and forth through pipette, 
then place a drop of the mixture near the end of each of 
the three slides. Make a thin smear by touching cigarette 
paper to the drop and pulling paper gently along the slide. 
Dry smear in the air, fix in 7% mercuric chloride for one 
minute, wash in water and stain in alkaline methylene 
blue 5 minutes. 

To count the bacteria examine the smears with oil im- 
mersion lens, counting the red blood cells and the bac- 
teria in about 25 fields on each slide, usually a total of 
about 300 or 400 red cells. Put the number of red cells 
counted in each field in one column and the number of 
bacteria counted in each corresponding field in another 
column, and sum up each separately. A small circle about 
Yz inch in diameter drawn on the lower glass, of the eye 
piece of the microscope with a pen or glass pencil forms 
a convenient small field in which all the cells can be easily 
and quickly counted. Well and evenly spread portions 
of the smears should be used in counting. 

To calculate the number of bacteria per c.c. in the emul- 
sion the following proportion is determined, it being known 
there are 5 million red cells per cmm. of blood. 

Number of red cells counted 5 million 



Number of bacteria counted x 

x= number of bacteria per cmm. multiplied by 1000= 
number of bacteria per c.c. 

Diluting and Bottling the Vaccine. — 

Bacterial vaccines of staphylococci are usually diluted to 
contain 1000 million bacteria per c.c, streptococci 200 mil- 



MANUAL OF LABORATORY DIAGNOSIS 125 

lion per c.c, and most bacilli 500 million per c.c. In dilut- 
ing calculate the total number of bacteria which will be 
present in the vaccine; for example there will be 25,000 
million staphylococci in a vaccine bottle containing 25 c.c. 
of vaccine with 1000 million bacteria per c.c. If the bac- 
terial emulsion as prepared contains 5000 million bac- 
teria per c.c, 5 c.c. of this emulsion must be added to 
20 c.c. of salt solution in order to have 25 c.c. of vaccine 
with 1000 million bacteria per c.c. 

It is convenient to have on hand amber bottles contain- 
ing 25 c.c. of normal salt solution cotton plugged and ster- 
ilized in the autoclav. Use a glass Luer syringe (10 c.c.) 
in making the dilution, sterilizing the syringe by drawing 
ether back and forth through the needle and the syringe 
a few times. Having calculated the number of c.c. of the 
bacterial emulsion required in the complete vaccine, that 
number of c.c. of salt solution is withdrawn from a bottle 
containing 25 c.c. with the sterile syringe and replaced 
by a corresponding number of c.c. of the emulsion. Add 
as antiseptic J / 2 c.c. of a 5% solution of carbolic acid to 
the 25 c.c. of vaccine. Fit rubber top previously immersed 
in 7% mercuric chloride for 5 minutes on bottle and place 
rubber band tightly just below rim of bottle. It is prac- 
tical to use heavy surgeon's rubber finger cots, cutting off 
the finger portion so as to leave about 1 inch of tip. 

To determine the sterility of the vaccine, shake the bottle, 
touch rubber top with lysol, withdraw from inverted bottle 
about y 2 c.c. of vaccine into sterile Luer syringe. Inoculate 
this into a tube of agar or blood serum and incubate 24 
hours. If the vaccine has been prepared from an anaero- 
bic organism requiring special media for growth, use the 
same conditions in planting out vaccine as were necessary 
in growing the bacteria in the first place. 



INDEX 



Abel bacillus, 112 

Abscess of lung, sputum in, 95 

Acchroodextrin, test for, 72 

in stomach contents, 69 
Acetonuria, Gunning's test, 53 
Acidosis, 53 
Acid, diacetic in urine, 53, 54 

hydrochloric, combined, 69. 
70, 73 
free, 69, 70. 73. 74 

lactic, 71, 74 
Acidity, total of gastric con- 
tents, 70, 73 

of urine, 45, 46 
Aestivo-autumnal parasite, 27 
Agglutination. See Widal re- 
action 
Albumiuria, 48 

Esbach's test, 51 

Heller's test. 49 

nitric acid test. 49 

occurrence, 48 

Purdy's qualitative test, 50 

Purdy's quantitative test, 50 

Robert's test, 50 

Tsuchiya's test, 51 
Alizarin 70, 71 
Amboceptor, 34 

titration of, 36 
Anaerobic cultures, 101 

making of, 101 
Anemia, blood in secondary, 22 
pernicious, 24 
chlorosis, 23 
Anilin-gentian violet, 102 
Anisocytosis, 19 
Antigen, 34 

titration of, 37 
Ascaris lumbricoides, 82 
Ascitic glucose agar, 100 
Asthma, sputum in, 95 



B 

Bacillus, Abel, 112 

anthracis, 117 

aerogenes capsulatus, 118 

coli, 113, 114, 115 

diphtheriae, 109 

diphtheroid, 109 

Ducrey, 112 

dysenteriae, 113, 114 

Friedlanders, 112 

influenzae, 111 

Koch-Weeks, 112, 120 

Morax-Axenfeld, 120 

mucosus capsulatus, 112 

ozenae, 112 

paratyphosus, 113, 114 

Perez, 112 

pertussus, 112 

proteus, 115 

pyocyaneus, 116 

tetani, 116 

tuberculosis, 108 

typhosus, 113. 114, 115 

Welchi, 118 

Xerosis, 111, 120 
Bacterial examination of 

blood, 98, 115 

conjunctival secretion, 120 

feces, 98, 115 

glands, 98 

sputum, 98 

tonsils, 98 

urine, 98, 115 
Bacterial smears, making of, 99 
Bacterial vaccines, 121 
Basic stippling, 19 
Basophiles, 16, 22 
Bence Jones proteid, 48, 49 
Bile, Gmelin's test, 59 

Smith-Rosen test, 59 
Bile medium for typhoid cul- 
tures, 100 



127 



128 



INDEX 



Blood, 
agar, 100 
casts, 65 

coagulation time, 17 
color index, 14 
culture, 9$ 

eosinophilia, occurrence of, 21 
erythrocyte, 

counting of, 10 
variation in, being nucle- 
ated, 19 
number, 18 
shape, 19 
size, 19 
staining, 19 
film, making of, 14 

staining, 15 
guaiac test for, 72, 79 
hemoglobin, 9 
in chlorosis, 23 
feces, 79, 80 
leukemia, 25 
lymphatic leukemia, 25 
myeloid leukemia, 25 
pernicious anemia, 24 - 
secondary anemia, 22 
sputum, 93 

stomach contents, 72, 74 
urine, 63 
leucocytes, counting of, 13 
differential count, 17 
percentage, 16 
varieties, 16 
leucocytosis, occurrence, 21 
pathologically, 21 
physiologically, 21 
lymphocytosis, occurrence, 21 
pathologically, 21 
physiologically, 21 
malarial parasites, methods of 
examination for, 27 
obtaining of, 9 
pathology, 18 



polynucleosis, 21 

serum, Loefflers, 100 

stain, 15 

typhoid bacillus in, 115 

Wassermann reaction, 31 

Weber's test for, 72, 79 

Widal reaction, 29 

Wright's stain, 15 
Blood agar, 100 
Blood cast, 65 

Blood cells. See Erythrocyte. 
Blood culture, 98 
Boaz Oppler bacillus, 75, 76 
Bronchiectasis, sputum in, 95 
Bronchitis, sputum in, 94 



Calcium oxalate crystals in 

urine, 61 
Carbol iuchsin, 103 
Carbonates in urine, 61 
Casts, 64 

blood, 65 

epithelial, 6 C 

fatty, 65 

granular, 64 

hyalin, 64 

pus, 65 

waxy, 64 
Cerebrospinal fluid. See Spinal 

fluid. 
Chlorosis, blood in, 23 
Coagulation time of blood, 17 

determination of, 17 
Colon-typhoid group of bacilli, 

113 
Colostrum corpuscles, 85 
Complement, 34 

fixation, 32 

fixation for Gonorrhea, 41 

titration of, 36 
Conjunctival secretion, 

bacteriology of, 120 



INDEX 



129 



Culture media, 99 
Cystin in urine, 61 
Cytology of spinal fluid, 87 



Degree of acidity of gastric 
contents, 71 

Diacetic acid in urine, 53 
Gerhardt's test, 54 

Diazo-reaction, 59 

Dimethylamido-azobenzol indi- 
cator, 71 

Diphtheria, diagnosis, 111 

Doremus' ureometer, 54 



Ehrlich's diazo reaction, 59 
Endo-medium, 100 
Eosinophile leucocyte, 16 
Eosinophilia, occurrence, 21 
Epithelial cast, 65 
Erythrocyte, counting of 10 
variation in being nucleated. 
19 

number, 18 

shape. 19 

size, 19 

staining, 19 
in urine. 63 
Erythrodextrin. 69. 72 
Ewald test breakfast, 69 



Fat in feces, 81 

in milk, 85 
Fatty cast. 65 
Feces, blood in. 80. 78, 79 
color of, 77 
examination of, 77 
bacterial, 115 
chemical, 79 
macroscopical, 77 
microscopical, 77 



fat in, 81 

food remnants in, 77 
uncus in, 78, 79 
ova in, 78, 82 
parasites of, 78, 82 
pathological findings and sig- 
nificance of, 79 
stones in, 81 
vegetable detritus of, 78 
Friedlanders bacillus, 112 



Grangrene of the lung, sputum 

in, 95 
Gastric contents, 
blood in, 72, 74 
Boaz oppler bacillus in. 75, 76 
combined hydrochloric acid 

in, 71, 73. 
degree of acidity of, 71 
examination of, 69 
chemical, 70 
macroscopical, 69 
microscopical, 70 
ferments of, 69, 71, 74 
free hydrochloric acid of, 70, 

73, 74 
in chronic gastritis, 76 
gastric carcinoma, 76 
gastric ulcer, 76 
hyperacidity, 76 
lactic acid in, 71, 74 
normal after Ewald test 

breakfast, 73 
mucus in, 70, 74 
pathological variation from 

normal, 73 
proteid digestion, 69, 72, 74 
sarcinae in, 75 
test meals, 69 
titration of, 70 
total acidity of. 70. 73 
yeasts in. 75 



130 



IXDEX 



Gastritis, gastric contents in, 76 
Globulin in cerebrospinal fluid, 
88 

Ross-Jones test for, 88 
Glycosuria, 51 

Haines' test, 52 

Haines' quantitative test, 52 
Gmelin's test for bile, 59 
Gonococcus, 107 
Gonorrhea 

Complement fixation test for, 
41 
Gram negative diplococcus 

group, 107 
Granular cast, 64 



H 



Hayem's fluid, 10 
Hemoglobin, estimation of, 9 
Hemoglobinophilic group of 

bacilli, 111 
Hemolysis, 31 
Hemolytic system, 31 
Hook worm, 82 
Hyalin cast, 64 

Hydrochloric acid, free, 70. 71, 
73, 74 
combined, 71, 73 
titration of, 70 
Hyperacidity, gastric contents 

in, 76 
Hypobromite method of esti- 
mation of urea, 54 



Indican, excess, 57 
Obermayer's test, 57 

K 

Kelling's test for lactic acid, 71 



Lactic acid in gastric contents, 

74, 76 
Lactose in milk, 86 

in urine, 51 
Lange colloidal gold reaction, 
89 

diagnostic value, 90 
Leucin and tyrosin in urine, 61 
Leucocytes, counting of, 13 

differential count, 17 

percentage of each variety, 16 

varieties of, 16 
Leucocytosis, 20 
Leukemia, blood in, 25 
Loeffler's methylene blue, 101 

blood serum, 100 
Lymphatic leukemia, blood in, 

26 
Lymphocytes, small, 16 
Lymphocytosis, 21 



M 



Macrocyte, 19 
Malarial parasites, 27 

methods of examination, 27 

aestivo autumnal, 27 

quartan, 27 

tertian, 27 
Mast cell, 16 
Megaloblast, 19 
Meningococcus, 108 
Meningitis, spinal fluid in, 91 
Microblast, 19 
Milk, human, 

fat in, 85 

colostrum corpuscles of, 85 

composition of, 85 

lactose of, 86 

proteid of, 86 

reaction of, 85 

specific gravity of, 85 



INDEX 



131 



Morax-Axenfeld bacillus, 120 

Mucin in urine, 49 

Mucosus capsulatus group of 

bacilli. 112 
Mucus, in feces, 78. 79 

in gastric contents. 70, 74 
Myelocyte, 20 
Myeloid leukemia, blood in. 25 



N 



Normoblast. 19 



Rennin in gastric contents, 
test for, 71 

Ross-Jones test for globulin, 88 

Rudolpf's method, modification 
of, for determina- 
tion of coagulation 
time of blood. 17 

Ruhemann's method of estima- 
tion of uric acid. 56 



Ova in feces, 78, 82 
Oxyuris vermicularis. 82 
Oligocythaemia, 18 
Obermaver's test for indican, 57 



Parasites in feces. 78. 82 
Perez bacillus, 112 
Phenolphthalein, 70, 71 
Phosphates in urine, 61 
Pneumococcus, 104 
Pneumonia, sputum in, 94 
Poikilocytes. 19 
Polychromatophilia. 19 
Polycythemia, 19 
Polynucleosis, 20 
Proteid digestion in gastric 

contents, 74 
Proteid in milk, 86 
Pulmonary tuberculosis, sputum 

in, 94 
Pulmonary edema, sputum in. 

95 
Pus casts in urine, 65 
Pus cells in urine, 63 
Pus in feces, 80 
urine. 63 



Quartan malarial parasite, 27 



Sarciriae in gastric contents, 75 
Seat worm, 82 
Smears, making of, 99 
Smith-Rosen test for bile, 59 
Specific gravity of urine, 46 

of breast milk, 85 
Spinal fluid, 87 

bacteriological examination 

of, 87 
chemical examination of, 88 
cytology of, 87 
differential cell count of, 87 
globulin of, 87 
in acute meningitis, 91 

tubercular meningitis. 91 
syphilitic disease of nerv- 
ous system, 91 
Lange colloidal gold reac- 
tion. 89 
normal. 87 
Ross-Jones test for globulin. 

88 
total cell count of, 88 
Wassermann. 40 
Sputum, 
bacteria of, 94 
character of. 93 
color of, 93 
consistency of, 93 
examination of. 
macroscopic, 93 



132 



IXDEX 



microscopic, 93 
in abcess of lung, 95 
acute bronchitis, 94 
acute lobar pneumonia. 94 
asthma, 95 
bronchiectasis, 95 
chronic bronchitis, 95 
gangrene, 95 
pulmonary edema, 95 
pulmonary tuberculosis, 94 
tubercle bacillus in, 109 
unstained, 93 
Staines and methods of stain- 
ing, 101 
Gram, 102 
Methylene blue, 101 
Wrights, 15 
Ziehl-Neelsen, 103 
Staphylococci, 103 
Starch digestion in stomach 

contents, 74 
Stomach contents. See Gastric 

contents. 
Stomach worm, 82 
Stones in feces, 81 
Stronglyoides intestinalis, 82 
Streptococci, 104 



Tallquist-hemoglo'bin scale. 10 
Test meals, 69 
Tertian malarial parasite. 27 
Tonsils, bacterial examination 

of, 98 
Topfer's reagent, 71 
Transitional leucocytes, 16 
Trichina spiralis, 82 
Trichomycetes, 118 
Trichocephalus trichiuris, 82 
Tubercle bacillus, in sputum, 109 

in urine, 109 

staining of, 103 
Typhoid bacillus, in blood, 115 



feces, 115 

urine, 115 
Typhoid fever, 

diazo reaction in, 59 
Widal reaction in, 31 

U 

Ulcer, gastric, gastric contents 

in, 76 
Uncinaria duodenalis, 82 
Urates in urine, 60 
Urea, decreased, 54 
increased, 54 

estimation of by hypobro- 
mite method, 54 
Uric acid, decreased, 56 

increased, 56 

estimation, Ruhemann's 
method, 56 

crystals, 60 
Urine, 

acidity, 46 

acidosis, 53 

albuminuria, 48 

appearance, 44 

amount, 43 

bacterial examination of, 98 

bile, 58 

blood cells, 63 

blood cast, 65 

calcium oxalate, 61 

carbonates, 61 

casts, 64 

chemical composition, normal, 
47 

color, 45 

cylindroids, 65 

cystin, 61 

diazo reaction, 59 

epithelial casts, 65 

epithelial cells, 62 

fatty casts, 65 



INDEX 



133 



glycosuria, 51 
granular casts, 64 
hyalin casts, 64 
in acute nephritis, 67 

chronic parenchymatous 
nephritis, 67 

diabetes mellitus, 67 

diabetes insipidus, 67 

renal calculus, 67 

tuberculosis of kidney, 67 
indican, 57 

leucin and tyrosin. 61 
obtaining, 43 
phosphates. 61 
pus casts, 65 
pus cells, 63 
reaction. 45 
red cells, 63 

sediment, unorganized in acid 
acid urine, 60 
in alkaline urine, 61 
organized, 62 
specific gravity, 46 
total solids, 47 
urates, 60 
urea, 54 
uric acid, 56 
waxy casts. 64 
yeast. 66 

V 

Vaccines, preparation of. 121 



\ incents angina. 121 

W 

Wassermann test, 31 

amboceptor, 34. 36 

antigen, 34. 37 

complement, 34, 36 

complement fixation, 32 

diagnostic value, 40 

hemolysis, 31 

hemolytic system, 31 

patients serum. 35 

sheeps corpuscles, 33 

technic, 38 

titration of reagent, 36 

with cerebrospinal fluid, 40 
Waxy cast, 64 

Weber's test for blood, 72, 79 
Whip worm, 82 
Widal reaction for typhoid, 29 
Wright's blood stain, 15 



Yeast in stomach contents, 75 



Ziehl-Neelsen method for stain- 
ing tubercle bacil- 
lus, 103 



FORM FOR REPORT BLANKS 



NO. 



Patient 



Date 



Physician 



Haemoglobin 



BLOOD EXAMINATION 



Erythrocyte Count 



Leucocyte Count 



Color Index 



STAINED FILM 
DIFFERENTIAL LEUCOCYTE COUNT 
Polymorphonuclear Neutrophiles Poikilocytosis 



ERYTHROCYTES 



Small Mononuclears 


Anisocytosis 


*_arge Mononuclears 


Polychromatophilia 


Transitionals 


Nucleated Reds Per 100 Leucocytes 


Eosinophiles 


Normoblasts 


Basophiles 


Megaloblasts 


Myelocytes 


Microblasts 




Parasites 


Coagulation Time 


Widal Reaction 


Opsonic Index 



Serum Tests 

Wassermann 



Complement Fixation Test for Gonorrhoea 



FORM FOR REPORT BLANKS 



Date 



Physician 






URINALYSIS 



QUALITATIVE 



Color 



QUANTITATIVE 
Quantity in 24 Hours 



Reaction 


Total Acidity 


Specific Gravity 


Total Solids 


Albumin 


Quantity 


Sugar 


Quantity 


Indican 


Urea 


Bile 


Uric Acid 


Rlnod 


Chlorides 


Acetone 


Sulohates 


Diacetic Acid 


Phosphates 


Diazo Reaction 


Ammonia 


Functional Kidney Test 


MICROSCOPIC: 
Casts 


Cylindroids 


Blood 


Pus 


Crystals 


AmorDhous Deposits 


EDithelial Cells 



FORM FOR REPORT BLANKS 



Patient 



Date 



Physician 



Test Meal 



EXAMINATION OF STOMACH CONTENTS 
Withdrawn in 



CHEMICAL 



Hrs. 



Quantity 


Rel. Amt. Liquid 


Odor 


Food Particles 


Color 


Mucus 



Total Acidity 


Starch 


Free HCL 


Erythrodextrin 


Combined HCL 


Acchroodextrin 


Free Acids and Salts 


Peptone 


Organic Acids and Salts 


Bile 


Lactic Acid 


Blood 


Ferments 


Pus 



Bacteria 



MICROSCOPIC 
Pus 



Yeast 



Blood 



Sarcinae 


Epithelium 


Oppler Boas B 


Mucosa 


Food Remnants From Former Meals 





FORM FOR REPORT CLANKS 

No. Patient Date 

Physician 

EXAMINATION OF FECES 
Color 



Consistency 


Reaction 


Undigested 


Food 


Connective 


Tissue 


Stones 


Fat 


Blood 




Bile 



MICROSCOPIC: 
Muscle Fibre 



Free Starch Granules 



Neutral Fat 



Crystal: 



Pus 



Parasites 



FORM FOR REPORT BLANKS 



No. 



Patient 



Date 



Physician 



Amount 



CEREBROSP NAL FLUID 



Appearance 



Total Cell Count 



Differential Cell Count 



Globulin Tests 



Fehling's Solution Reduction 



Lange's Colloidal Gold Test 



Wassermann 



Bacteriological Examination 



Stained Smears from Sediment 



Cultures 



LIBRARY OF CONGRESS 




007 635 065 6 



